Ultra-potent neutralization of cytokines by multispecific antibodies and uses thereof

ABSTRACT

The present invention provides multispecific antibodies, and antigen binding fragments thereof, that potently neutralize a cytokine and that may thus be useful in the prevention and/or treatment of inflammatory and/or autoimmune diseases. In particular, the present invention provides a multispecific antibody, or an antigen binding fragment thereof, comprising at least two different domains specifically binding to at least two different, non-overlapping sites in a cytokine and an Fc moiety. The invention also relates to nucleic acids that encode such antibodies and antibody fragments and immortalized B cells and cultured plasma cells that produce such antibodies and antibody fragments. In addition, the invention relates to the use of the antibodies and antibody fragments of the invention in screening methods as well as in the diagnosis, prophylaxis and treatment of inflammatory and/or autoimmune diseases.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 480369.403D1_SEQUENCE_LISTING.txt. The text file is 209 KB, was created on Jan. 9, 2020, and is being submitted electronically via EFS-Web.

The present invention relates to multispecific anti-cytokine, preferably anti-GM-CSF, antibodies, and antigen binding fragments thereof, that potently neutralize cytokines, in particular GM-CSF. The multispecific anti-cytokine, preferably anti-GM-CSF, antibodies of the present invention comprise at least two different domains specifically binding to at least two different, non-overlapping sites on a single cytokine molecule. The invention also relates to nucleic acids that encode and immortalized B cells and cultured plasma cells that produce such antibodies and antibody fragments. In addition, the invention relates to the use of the antibodies and antibody fragments of the invention in screening methods as well as in the diagnosis, prophylaxis and treatment of diseases, in particular inflammatory and autoimmune diseases.

Cytokines are a family of immune-modulatory molecules that are secreted by a variety of cells and act locally or systemically on other cells. Most cytokines have immune-modulatory activity, being involved in the control of different types of inflammatory processes and mechanisms of host defense. Cytokines include chemokines, interferons, interleukins, lymphokines, tumour necrosis factor, monokines and colony stimulating factors. Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells; a given cytokine may be produced by more than one type of cell.

The cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) is a monomeric protein of 127 amino acids with a molecular weight ranging between 14 kDa and 35 kDa depending on the variable degree of glycosylation. Non-glycosylated and glycosylated GM-CSF show similar activity in vitro (Cebon, J., Nicola, N., Ward, M., Gardner, I., Dempsey, P., Layton, J., Diihrsen, U., Burgess, A. W., Nice, E., and Morstyn, G. (1990). Granulocyte-macrophage colony stimulating factor from human lymphocytes. The effect of glycosylation on receptor binding and biological activity. J. Biol. Chem. 265, 4483-4491). GM-CSF exerts its biological activity by binding to its receptor (Hansen, G., Hercus, T. R., McClure, B. J., Stomski, F. C., Dottore, M., Powell, J., Ramshaw, H., Woodcock, J. M., Xu, Y., Guthridge, M., et al. (2008). The structure of the GM-CSF receptor complex reveals a distinct mode of cytokine receptor activation. Cell 134, 496-507), which is expressed on the cell surface of myeloid cells and endothelial cells but absent on lymphocytes. The receptor is heterodimeric and is composed of alpha and beta subunits. The alpha subunit binds GM-CSF with nanomolar affinity. The beta subunit is also part of the interleukin-3 and interleukin-5 receptor complexes and, in association with the GM-CSF receptor alpha subunit and GM-CSF, leads to the formation of a multimeric complex where GM-CSF is bound with picomolar binding affinity.

GM-CSF can be expressed by a variety of cell types including T lymphocytes, macrophages, NK cell, mast cells, endothelial cells, fibroblasts and some malignant cells (Gasson, J. C. (1991). Molecular physiology of granulocyte-macrophage colony-stimulating factor. Blood 77, 1131-1145; Hamilton, J. A., and Anderson, G. P. (2004). GM-CSF Biology. Growth Factors 22, 225-231; Sergeeva, A., Ono, Y., Rios, R., and Molldrem, J. J. (2008). High titer autoantibodies to GM-CSF in patients with AML, CML and MDS are associated with active disease. Leukemia 22, 783-790). GM-CSF acts as a growth factor on hemopoietic precursor cells to produce granulocytes and monocytes. It is also essential for the development of microglia and for the function of alveolar macrophages. In addition, GM-CSF has also a variety of pro-inflammatory effects on cells of the immune system expressing the GM-CSF receptor. The most important of these functions is the activation of monocytes, macrophages and granulocytes in several inflammatory and autoimmune diseases (Hamilton, J. A. (2002). GM-CSF in inflammation and autoimmunity. Trends Immunol 23, 403-408), which in turn result in the production of other cytokines and chemokines, matrix degrading proteases, increased HLA expression and increased expression of adhesion molecules or CC-chemokines receptors. GM-CSF can also synergize with other inflammatory factors like other cytokines or LPS (Parajuli, B., Sonobe, Y., Kawanokuchi, J., Doi, Y., Noda, M., Takeuchi, H., Mizuno, T., and Suzumura, A. (2012). GM-CSF increases LPS-induced production of proinflammatory mediators via upregulation of TLR4 and CD14 in murine microglia. J Neuroinflammation 9, 268). Taken together, GM-CSF is thus part of the immune/inflammatory cascade.

GM-CSF can therefore be considered as a target for anti-inflammatory and autoimmune therapies. Chronic and acute inflammatory and/or autoimmune diseases such as for example rheumatoid arthritis (RA), multiple sclerosis (MS), Crohn's disease, psoriasis, asthma, atopic dermatitis or shock may benefit from GM-CSF neutralization and the consequent pro-inflammatory cascade. For example in MS elevated levels of GM-CSF correlate with the active phase of MS (McQualter, J. L., Darwiche, R., Ewing, C., Onuki, M., Kay, T. W., Hamilton, J. A., Reid, H. H., and Bernard, C. C. (2001). Granulocyte macrophage colony-stimulating factor: a new putative therapeutic target in multiple sclerosis. J Exp Med 194, 873-882; Noster, R., Riedel, R., Mashreghi, M.-F., Radbruch, H., Harms, L., Haftmann, C., Chang, H.-D., Radbruch, A., and Zielinski, C. E. (2014). IL-17 and GM-CSF expression are antagonistically regulated by human T helper cells. Sci Transl Med 6, 241ra80-241ra80) and GM-CSF deficient mice fail to develop disease in the experimental autoimmune encephalomyelitis (EAE) murine model for multiple sclerosis (McQualter, J. L., Darwiche, R., Ewing, C., Onuki, M., Kay, T. W., Hamilton, J. A., Reid, H. H., and Bernard, C. C. (2001). Granulocyte macrophage colony-stimulating factor: a new putative therapeutic target in multiple sclerosis. J Exp Med 194, 873-882).

In asthma, increased levels of GM-CSF are found in the airways of asthmatic patients (Broide, D. H., and Firestein, G. S. (1991). Endobronchial allergen challenge in asthma. Demonstration of cellular source of granulocyte macrophage colony-stimulating factor by in situ hybridization. J Clin Invest 88, 1048-1053; Sousa, A. R., Poston, R. N., Lane, S. J., Nakhosteen, J. A., and Lee, T. H. (1993). Detection of GM-CSF in asthmatic bronchial epithelium and decrease by inhaled corticosteroids. Am. Rev. Respir. Dis. 147, 1557-1561). Indeed, GM-CSF in synergy with IL-5 promotes the differentiation, activation and survival of eosinophils (Yamashita, N., Tashimo, H., Ishida, H., Kaneko, F., Nakano, J., Kato, H., Hirai, K., Horiuchi, T., and Ohta, K. (2002). Attenuation of airway hyperresponsiveness in a murine asthma model by neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF). Cellular Immunology 219, 92-97). The usefulness of GM-CSF neutralizing antibodies was demonstrated in a mouse model of asthma where their administration led to significant reduction of airway hyperresponsiveness and inflammation (Yamashita, N., Tashimo, H., Ishida, H., Kaneko, F., Nakano, J., Kato, H., Hirai, K., Horiuchi, T., and Ohta, K. (2002). Attenuation of airway hyperresponsiveness in a murine asthma model by neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF). Cellular Immunology 219, 92-97)).

In lung diseases, GM-CSF has also a role where high neutrophil numbers, protease induction, and TNF-alpha overproduction are believed to be central agents in disease pathogenesis, like occupational lung diseases caused by LPS-containing bioaerosols. Indeed, in a mouse model LPS-dependent inflammation of the lung was reduced by using a GM-CSF neutralizing antibody (Bozinovski, S., Jones, J., Beavitt, S.-J., Cook, A. D., Hamilton, J. A., and Anderson, G. P. (2004). Innate immune responses to LPS in mouse lung are suppressed and reversed by neutralization of GM-CSF via repression of TLR-4. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L877-L885).

In RA, multiple groups have measured high levels of GM-CSF in the synovial joint fluids (Alvaro-Gracia, J. M., Zvaifler, N.J., Brown, C. B., Kaushansky, K., and Firestein, G. S. (1991). Cytokines in chronic inflammatory arthritis. VI. Analysis of the synovial cells involved in granulocyte-macrophage colony-stimulating factor production and gene expression in rheumatoid arthritis and its regulation by IL-1 and tumor necrosis factor-alpha. J Immunol 146, 3365-3371; Haworth, C., Brennan, F. M., Chantry, D., Turner, M., Maini, R. N., and Feldmann, M. (1991). Expression of granulocyte-macrophage colony-stimulating factor in rheumatoid arthritis: regulation by tumor necrosis factor-alpha. Eur J Immunol 21, 2575-2579; Fiehn, C., Wermann, M., Pezzutto, A., Hufner, M., and Heilig, B. (1992). [Plasma GM-CSF concentrations in rheumatoid arthritis, systemic lupus erythematosus and spondyloarthropathy]. Z Rheumatol 51, 121-126) and treatment with recombinant GM-CSF after chemotherapy was shown to cause flares of RA (de Vries, E. G., Willemse, P. H., Biesma, B., Stern, A. C., Limburg, P. C., and Vellenga, E. (1991). Flare-up of rheumatoid arthritis during GM-CSF treatment after chemotherapy. The Lancet 338, 517-518). The therapeutic potential of GM-CSF neutralizing antibodies in RA was suggested from their efficacy in a collagen-induced arthritis model in mice (Cook, A. D., Braine, E. L., Campbell, I. K., Rich, M. J., and Hamilton, J. A. (2001). Blockade of collagen-induced arthritis post-onset by antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF): requirement for GM-CSF in the effector phase of disease. Arthritis Res. 3, 293-298; Cornish, A. L., Campbell, I. K., McKenzie, B. S., Chatfield, S., and Wicks, I. P. (2009). G-CSF and GM-CSF as therapeutic targets in rheumatoid arthritis. Nat Rev Rheumatol 5, 554-559).

Antibodies able to neutralize GM-CSF may thus represent new effective preventions and/or therapies for inflammatory and/or autoimmune diseases such as MS, RA and other autoimmune and inflammatory diseases. In principle, cytokine neutralization can be achieved by an antibody that binds to its target soluble cytokine or to the cytokine receptor displayed on the cell's membrane. MOR103 and Namilumab are two phage-derived human monoclonal antibodies that neutralizes GM-CSF and that are being developed as therapeutics in RA and MS (Steidl, S., Ratsch, O., Brocks, B., Dürr, M., and Thomassen-Wolf, E. (2008). In vitro affinity maturation of human GM-CSF antibodies by targeted CDR-diversification. Mol Immunol 46, 135-144; Krinner, E.-M., Raum, T., Petsch, S., Bruckmaier, S., Schuster, I., Petersen, L., Cierpka, R., Abebe, D., Molhoj, M., Wolf, A., et al. (2007). A human monoclonal IgG1 potently neutralizing the pro-inflammatory cytokine GM-CSF. Mol Immunol 44, 916-925; Behrens, F., Tak, P. P., Ostergaard, M., Stoilov, R., Wiland, P., Huizinga, T. W., Berenfus, V. Y., Vladeva, S., Rech, J., Rubbert-Roth, A., et al. (2014). MOR103, a human monoclonal antibody to granulocyte-macrophage colony-stimulating factor, in the treatment of patients with moderate rheumatoid arthritis: results of a phase Ib/IIa randomised, double-blind, placebo-controlled, dose-escalation trial. Ann Rheum Dis) and mavrilimumab is a human monoclonal antibody targeting GM-CSF receptor-alpha under development in RA patients (Burmester, G. R., Feist, E., Sleeman, M. A., Wang, B., White, B., and Magrini, F. (2011). Mavrilimumab, a human monoclonal antibody targeting GM-CSF receptor-α, in subjects with rheumatoid arthritis: a randomised, double-blind, placebo-controlled, phase I, first-in-human study. Ann Rheum Dis 70, 1542-1549).

However, single GM-CSF neutralizing antibodies lead in vivo to the accumulation of a large pool of long-lived GM-CSF that is still able to dissociate and trigger the receptor and that could be the basis of the enhancing activity of monoclonal antibodies to common gamma-chain cytokines in vivo (Boyman, O., Kovar, M., Rubinstein, M. P., Surh, C. D., and Sprent, J. (2006). Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science 311, 1924-1927).

In view of the above, it is an object of the present invention to provide an antibody, which neutralizes cytokines, in particular GM-CSF, more potently and efficiently than the antibodies presently available. Such an antibody can be used at lower doses, thereby reducing the risks of side effects and saving costs. Moreover, it is an object of the present invention to provide an antibody, which neutralizes cytokines, in particular GM-CSF, but which does not result in accumulation of a large pool of long-lived cytokines, in particular GM-CSF, which is still able to dissociate and to trigger their receptor. Taken together, it is thus the object of the present invention to provide improved antibodies, or antigen binding fragments thereof, as well as related nucleic acid molecules, vectors and cells and pharmaceutical compositions, which overcome the disadvantages of the prior art by a cost-effective and straight-forward approach.

The object underlying the present invention is solved by the claimed subject matter.

Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the term “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated member, integer or step but not the exclusion of any other non-stated member, integer or step. The term “consist of” is a particular embodiment of the term “comprise”, wherein any other non-stated member, integer or step is excluded. In the context of the present invention, the term “comprise” encompasses the term “consist of”. The term “comprising” thus encompasses “including” as well as “consisting” e.g., a composition “comprising” X may consist exclusively of X or may include something additional e.g., X+Y.

The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

The word “substantially” does not exclude “completely” e.g., a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

The term “about” in relation to a numerical value x means x±10%.

The term “disease” as used herein is intended to be generally synonymous, and is used interchangeably with, the terms “disorder” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.

As used herein, reference to “treatment” of a subject or patient is intended to include prevention, prophylaxis, attenuation, amelioration and therapy. The terms “subject” or “patient” are used interchangeably herein to mean all mammals including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits. In one embodiment, the patient is a human.

As used herein, the term “antibody” encompasses various forms of antibodies, preferably monoclonal antibodies including but not being limited to whole antibodies, antibody fragments, human antibodies, chimeric antibodies, humanized antibodies and genetically engineered antibodies (variant or mutant antibodies) as long as the characteristic properties according to the invention are retained. Especially preferred are human or humanized monoclonal antibodies, especially as recombinant human antibodies.

The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 3340). Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95). The term “human antibody” as used herein also comprises such antibodies which are modified, e.g. in the variable region to generate the properties according to the invention.

As used herein, the term “recombinant human antibody” is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as for example a CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the V_(H) and V_(L) regions of the recombinant antibodies are sequences that, while derived from and related to human germ line V_(H) and V_(L) sequences, may not naturally exist within the human antibody germ line repertoire in vivo.

As used herein, the terms “antigen binding fragment,” “fragment,” and “antibody fragment” are used interchangeably to refer to any fragment of an antibody of the invention that retains the specific binding activity of the antibody according to the invention and the Fc moiety. Examples of antibody fragments include, but are not limited to, a single chain antibody, Fab, Fab′, F(ab′)₂, Fv or scFv. Fragments of the antibodies of the invention can be obtained from the antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of antibodies can be obtained by cloning and expression of part of the sequences of the heavy and/or light chains. “Fragments” include, but are not limited to, Fab, Fab′, F(ab′)2 and Fv fragments. The invention also encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of an antibody of the invention. For example, the invention includes a scFv comprising the CDRs from an antibody of the invention. Also included are heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, as well as single chain antibodies, e.g., single chain Fv in which the heavy and light chain variable domains are joined by a peptide linker. Antibody fragments of the invention may impart monovalent or multivalent interactions and be contained in a variety of structures as described above. For instance, scFv molecules may be synthesized to create a trivalent “triabody” or a tetravalent “tetrabody.” The scFv molecules may include a domain of the Fc region resulting in bivalent minibodies. In addition, the sequences of the invention may be a component of multispecific molecules in which the sequences of the invention target the epitopes of the invention and other regions of the molecule bind to other targets. Exemplary molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies (Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136). Although the specification, including the claims, may, in some places, refer explicitly to antigen binding fragment(s), antibody fragment(s), variant(s) and/or derivative(s) of antibodies, it is understood that the term “antibody” or “antibody of the invention” includes all categories of antibodies, namely, antigen binding fragment(s), antibody fragment(s), variant(s) and derivative(s) of antibodies. Further, the term “antibody” as used herein includes both antibodies and antigen binding fragments thereof.

As used herein, a “neutralizing antibody” is one that can neutralize, i.e., prevent, inhibit, reduce, impede or interfere with, the ability of a pathogen to initiate and/or perpetuate an infection in a host. The terms “neutralizing antibody” and “an antibody that neutralizes” or “antibodies that neutralize” are used interchangeably herein. These antibodies can be used alone, or in combination, as prophylactic or therapeutic agents upon appropriate formulation, in association with active vaccination, as a diagnostic tool, or as a production tool as described herein.

As used herein, the term “variable region” (variable region of a light chain (V_(L)), variable region of a heavy chain (V_(H))) denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen. In a native antibody, the domains of variable human light and heavy chains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three “hypervariable regions” (or complementarity determining regions, CDRs). The framework regions adopt a β-sheet conformation and the CDRs may form loops connecting the β-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.

As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from the “complementarity determining regions” or “CDRs”. “Framework” or “FR” regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chains of a native antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs on each chain are separated by such framework amino acids. Especially, CDR3 of the heavy chain is the region which contributes most to antigen binding. CDR and FR regions are determined according to the standard definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

As used herein, the term “constant domains” refers to domains of an antibody which are not involved directly in binding an antibody to an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins are divided in the classes: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses, e.g. IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called α, ε, γ, and □, respectively. The antibodies according to the invention are preferably of IgG type.

As used herein, the term “nucleic acid or nucleic acid molecule” is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.

As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny. Thus, the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

Doses are often expressed in relation to the bodyweight. Thus, a dose which is expressed as [g, mg, or other unit]/kg (or g, mg etc.) usually refers to [g, mg, or other unit] “per kg (or g, mg etc.) bodyweight”, even if the term “bodyweight” is not explicitly mentioned.

The term “specifically binding” and similar reference does not encompass non-specific sticking.

The term “vaccine” as used herein is typically understood to be a prophylactic or therapeutic material providing at least one antigen, preferably an immunogen. The antigen or immunogen may be derived from any material that is suitable for vaccination. For example, the antigen or immunogen may be derived from a pathogen, such as from bacteria or virus particles etc., or from a tumor or cancerous tissue. The antigen or immunogen stimulates the body's adaptive immune system to provide an adaptive immune response. In particular, an “antigen” or an “immunogen” refers typically to a substance which may be recognized by the immune system, preferably by the adaptive immune system, and which is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be or may comprise a peptide or protein which may be presented by the MHC to T-cells.

As used herein, “sequence variant” refers to any alteration in a reference sequence, whereby a reference sequence is any of the sequences listed in the “Table of Sequences and SEQ ID Numbers” (sequence listing), i.e. SEQ ID NO: 1 to SEQ ID NO: 190. Thus, the term “sequence variant” includes nucleotide sequence variants and amino acid sequence variants. In particular, in a “sequence variant” the functionality (of the reference sequence) is preserved, i.e. the sequence variant is functional. A “sequence variant” as used herein typically has a sequence which is at least 70% identical to the reference sequence, preferably at least 80% identical to the reference sequence, more preferably at least 90% identical, even more preferably at least 95% identical, and particularly preferably at least 99% identical to the reference sequence.

Sequence identity is usually calculated with regard to the full length of the reference sequence (i.e. the sequence recited in the application). Percentage identity, as referred to herein, can be determined, for example, using BLAST using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap open penalty=11 and gap extension penalty=1].

As used herein, a “nucleotide sequence variant” has an altered sequence in which one or more of the nucleotides in the reference sequence is deleted, or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by the standard one-letter designation (A, C, G, or T). Due to the degeneracy of the genetic code, a “nucleotide sequence variant” can either result in a change in the respective reference amino acid sequence, i.e. in an “amino acid sequence variant” or not. Preferred sequence variants are such nucleotide sequence variants, which do not result in amino acid sequence variants (silent mutations), but other non-silent mutations are within the scope as well, in particular mutant nucleotide sequences, which result in an amino acid sequence, which is at least 70% identical to the reference sequence, preferably at least 80% identical to the reference sequence, more preferably at least 90% identical, even more preferably at least 95% identical, and particularly preferably at least 99% identical to the reference sequence.

As used herein, term “mutation” or “mutating” shall be understood to include physically making a mutation, e.g. in an nucleic acid sequence (e.g., by altering, e.g., by site-directed mutagenesis, a codon of a nucleic acid molecule encoding one amino acid to result in a codon encoding a different amino acid) or synthesizing a sequence variant (e.g., by knowing the nucleotide sequence of a nucleic acid molecule encoding a polypeptide and by designing the synthesis of a nucleic acid molecule comprising a nucleotide sequence encoding a variant of the polypeptide without the need for mutating one or more nucleotides of a nucleic acid molecule).

An “amino acid sequence variant” has an altered sequence in which one or more of the amino acids in the reference sequence is deleted or substituted, or one or more amino acids are inserted into the sequence of the reference amino acid sequence. As a result of the alterations, the amino acid sequence variant has an amino acid sequence which is at least 70% identical to the reference sequence, preferably at least 80% identical to the reference sequence, more preferably at least 90% identical, even more preferably at least 95% identical, and particularly preferably at least 99% identical to the reference sequence. Variant sequences which are at least 90% identical have no more than 10 alterations, i.e. any combination of deletions, insertions or substitutions, per 100 amino acids of the reference sequence.

In the context of peptides/proteins, a “linear sequence” or a “sequence” is the order of amino acids in a peptide/protein in an amino to carboxyl terminal direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the peptide/protein.

While it is possible to have non-conservative amino acid substitutions, it is preferred that the substitutions be conservative amino acid substitutions, in which the substituted amino acid has similar structural or chemical properties with the corresponding amino acid in the reference sequence. By way of example, conservative amino acid substitutions involve substitution of one aliphatic or hydrophobic amino acids, e.g. alanine, valine, leucine and isoleucine, with another; substitution of one hydroxyl-containing amino acid, e.g. serine and threonine, with another; substitution of one acidic residue, e.g. glutamic acid or aspartic acid, with another; replacement of one amide-containing residue, e.g. asparagine and glutamine, with another; replacement of one aromatic residue, e.g. phenylalanine and tyrosine, with another; replacement of one basic residue, e.g. lysine, arginine and histidine, with another; and replacement of one small amino acid, e.g., alanine, serine, threonine, methionine, and glycine, with another.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the fusion to the N- or C-terminus of an amino acid sequence to a reporter molecule or an enzyme.

Importantly, the sequence variants are functional sequence variants, i.e. the alterations in the sequence variants do not abolish the functionality of the respective reference sequence, in the present case, e.g., the functionality of a sequence of an antibody, or antigen binding fragment thereof, to bind to the same non-overlapping epitopes/sites of a cytokine, in particular GM-CSF, and/or to sufficiently neutralize the cytokine, in particular GM-CSF. Guidance in determining which nucleotides and amino acid residues, respectively, may be substituted, inserted or deleted without abolishing such functionality are found by using computer programs well known in the art.

As used herein, a nucleic acid sequence or an amino acid sequence “derived from” a designated nucleic acid, peptide, polypeptide or protein refers to the origin of the polypeptide. Preferably, the nucleic acid sequence or amino acid sequence which is derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, from which it is derived, whereby “essentially identical” includes sequence variants as defined above. Preferably, the nucleic acid sequence or amino acid sequence which is derived from a particular peptide or protein, is derived from the corresponding domain in the particular peptide or protein. Thereby, “corresponding” refers in particular to the same functionality. For example, a CDR (or CDRH1) amino acid sequence or nucleic acid sequence in a multispecific antibody, which is “derived from a (particular) monospecific antibody” is usually derived from the (corresponding) CDR (or CDRH1) amino acid sequence or nucleic acid sequence of this monospecific antibody (and not from another (non-corresponding) part of the monospecific antibody). “Corresponding” parts of peptides, proteins and nucleic acids are thus easily identifiable to one of ordinary skill in the art (e.g. CDR1 corresponds to CDR1, CDR2 corresponds to CDR2, etc.). Likewise, sequences “derived from” another sequence are usually easily identifiable to one of ordinary skill in the art as having its origin in the sequence.

Preferably, a nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be identical to the starting nucleic acid, peptide, polypeptide or protein (from which it is derived). However, a nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may also have one or more mutations relative to the starting nucleic acid, peptide, polypeptide or protein (from which it is derived), in particular a nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be a functional sequence variant as described above of the starting nucleic acid, peptide, polypeptide or protein (from which it is derived). For example, in a peptide/protein one or more amino acid residues may be substituted with other amino acid residues or one or more amino acid residue insertions or deletions may occur.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

It is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

The invention is based, amongst other findings, on the discovery that a cocktail of three antibodies binding to different non-overlapping sites in the same cytokine, in particular GM-CSF, is, on a weight basis, more potent than presently available antibodies MOR103 or Namilumab. In particular, when a cocktail of three antibodies is used, no accumulation of a large pool of long-lived GM-CSF occurs, but the antibodies form immune complexes that are rapidly degraded in vivo in an Fc-dependent fashion. This prompted the present inventors to design multispecific antibodies originated by the combination of the above described antibodies. Surprisingly, such multispecific antibodies have a greatly enhanced neutralizing activity not only compared to the presently available GM-CSF antibodies, but also compared to cocktail of antibodies, wherein each antibody is specific only for a single site of the cytokine. It is suggested that in the multispecific antibody constructs according to the present invention the cytokine, in particular GM-CSF, becomes irreversibly sequestered and no longer available for interaction with the receptor. These multispecific antibodies can be used at extremely low dosage to treat several cytokine-dependent, in particular GM-CSF-dependent, diseases such as autoimmune and inflammatory diseases.

Multispecific Antibodies, or Antigen Binding Fragments Thereof

In a first aspect, the present invention provides an isolated multispecific anti-cytokine, preferably anti-GM-CSF, antibody, or an antigen binding fragment thereof, comprising:

-   -   (a) at least two different epitope binding sites, each of them         specifically binding to an individual epitope of a cytokine,         whereby the individual epitopes of a cytokine to which the at         least two different epitope binding sites bind, are         non-overlapping epitopes; and     -   (b) an Fc moiety.

Such a multispecific anti-cytokine, preferably anti-GM-CSF, antibody, or antigen binding fragment thereof, according to the present invention typically potently neutralizes a cytokine, in particular a target effect of said cytokine, for example the multispecific antibody, or antigen binding fragment thereof, according to the present invention typically potently neutralizes a GM-CSF, in particular a target effect of said GM-CSF. Such neutralization may be assessed in a neutralization assay as known to the skilled person and described below.

Preferably, the multispecific anti-cytokine, preferably anti-GM-CSF, antibody, or antigen binding fragment thereof, according to the present invention comprises an amino acid sequence which is not naturally occurring.

As used herein, a “multispecific” antibody refers to an antibody, wherein a single antibody molecule can bind to at least two different epitopes, e.g. to at least two different, non-overlapping sites in a cytokine, in particular GM-CSF. In contrast to most known multispecific antibodies, which usually bind to at least two different epitopes on distinct molecules, a single molecule of the multispecific anti-cytokine, preferably anti-GM-CSF, antibody, or of the antigen binding fragment thereof, according to the present invention can bind to at least two different, non-overlapping sites on a single cytokine molecule, in particular on a single GM-CSF molecule. Thus, the multispecific antibody, or antigen binding fragment thereof, according to the present invention is “multispecific” in respect to a single cytokine (molecule).

Preferably, the multispecific anti-cytokine, preferably anti-GM-CSF, antibody, or antigen binding fragment thereof, according to the present invention is bispecific, trispecific, tetraspecific or pentaspecific, more preferably the antibody, or the antigen binding fragment thereof, is bispecific, trispecific or tetraspecific, even more preferably the antibody, or the antigen binding fragment thereof, is bispecific or trispecific, and particularly preferably the antibody, or the antigen binding fragment thereof, is trispecific. Thereby it is meant that the multispecific anti-cytokine, preferably anti-GM-CSF, antibody, or antigen binding fragment thereof, according to the present invention is bispecific, trispecific, tetraspecific or pentaspecific in respect to a single cytokine (molecule), in particular in respect to a single GM-CSF (molecule).

The epitopes, i.e. the sites in a cytokine, in particular in a single cytokine molecule, for example in GM-CSF, in particular in a single GM-CSF molecule, to which the antibodies of the invention bind, may be linear (continuous) or conformational (discontinuous). Preferably, the antibodies and antibody fragments of the invention bind a conformational epitope, more preferably the conformational epitope is present only under non-reducing conditions. However, antibodies and antibody fragments of the invention may also bind to a linear epitope, more preferably the linear epitope is present under both, non-reducing conditions and reducing conditions.

The antibody according to the present invention comprises at least two different epitope binding sites, each of them specifically binding to an individual epitope of a cytokine, whereby the individual epitopes of a cytokine to which the at least two different epitope binding sites bind, are non-overlapping epitopes, in particular non-overlapping epitopes of the primary sequence of the cytokine, e.g. GM-CSF. Importantly, the epitopes, i.e. the sites in a cytokine, in particular in a single cytokine molecule, for example in GM-CSF, in particular in a single GM-CSF molecule, to which the antibody of the invention binds, are different (i.e. not the same) and non-overlapping. In a single cytokine (e.g. GM-CSF) molecule the epitopes (also referred to as “sites”), to which the multispecific anti-cytokine, preferably anti-GM-CSF, antibody according to the present invention binds, may be arranged directly adjacently or may be separated, e.g. by a linker, by another antibody domain, etc., but according to the present invention the epitopes must not overlap. Thereby, non-overlapping means that no amino acid in the amino acid sequence of the cytokine (e.g. GM-CSF) is used in more than one epitope/site to which the antibody according to the invention binds. In other words, each amino acid in the amino acid sequence of the cytokine (e.g. GM-CSF) is either part of one single epitope to which the antibody according to the invention binds or is no part of any epitope to which the antibody according to the invention binds.

Accordingly, the antibody, or antigen binding fragment thereof, according to the present invention can also be used to map the epitopes of the cytokine (e.g. GM-CSF) to which they bind.

The antibody, or antigen binding fragment thereof, according to the present invention comprises at least two different domains for specifically binding to the at least two different, non-overlapping sites in a cytokine (e.g. GM-CSF). In other words, the antibody, or antigen binding fragment thereof, according to the present invention comprises at least one first domain specifically binding to a first site in a cytokine (e.g. GM-CSF) and at least one second domain, which is different from the at least one first domain and which specifically binds to a second site in a cytokine (e.g. GM-CSF), whereby the second site in the cytokine (e.g. GM-CSF) is different from and non-overlapping with the first site in the cytokine (e.g. GM-CSF).

Herein the domains of the antibody, which specifically bind to the cytokine (e.g. GM-CSF), may be also termed “binding domains”, “epitope binding domains” or “epitope binding sites”. Preferably, such an epitope binding site of the antibody comprises at least one, preferably three and more preferably six CDRs, which fulfill at least the minimal requirements for specifically binding to a distinct epitope (thus constituting an “epitope binding site”) and which may be for example derived from a monospecific antibody. Accordingly, it is more preferred if such an epitope binding site of the antibody comprises six CDRs forming together the epitope binding site, e.g. CDR1, CDR2, and CDR3 derived from a heavy chain of a monospecific antibody and CDR1, CDR2, and CDR3 derived from the corresponding light chain of the same monospecific antibody. Even more preferably, such an epitope binding site of the multispecific anti-cytokine, preferably anti-GM-CSF, antibody according to the present invention may comprise a heavy chain variable region and/or a (corresponding) light chain variable region, which may be for example derived from (the same) monospecific antibody.

In principle, each different epitope binding site of the multispecific anti-cytokine, preferably anti-GM-CSF, antibody, or of the antigen binding fragment thereof, according to the present invention may be present in the antibody one or more, e.g. two, three, four, five, six, or more times. For example, native IgG is bivalent and monospecific, because it contains two identical Fabs, both recognizing the same epitope. Thus, the multispecific antibody, or the antigen binding fragment thereof, according to the present invention is at least bivalent, i.e. in the case of two different epitope binding sites each occurring once in the antibody. Moreover, the multispecific antibody, or the antigen binding fragment thereof, according to the present invention may also be trivalent, e.g. in the case of three different epitope binding sites each occurring once or in the case of two different epitope binding sites, one occurring once and the other twice; tetravalent, e.g. in the case of four different epitope binding sites each occurring once or in the case of three different epitope binding sites, two occurring once each and the third one occurring twice or in the case of two different epitope binding sites each occurring twice or one occurring once and the other three times; pentavalent; hexavalent; heptavalent; octavalent; nonavalent; decavalent; undecavalent; dodecavalent; tridecavalent; tetradecavalent etc.

Preferably, each of the different epitope binding sites occurs twice in the antibody molecule according to the present invention. In other words, the antibody molecule according to the present invention comprises exactly two copies of each of the different domains specifically binding to at least two different, non-overlapping sites in a cytokine comprised by the antibody, or the antigen binding fragment thereof. Accordingly, the multispecific antibody, or the antigen binding fragment thereof, according to the present invention is preferably tetravalent, hexavalent, octavalent, decavalent, dodecavalent, tetradecavalent, etc., whereby the antibody molecule comprises exactly two copies of each of the different epitope binding sites. More preferably, the multispecific anti-cytokine, preferably anti-GM-CSF, antibody, or the antigen binding fragment thereof, according to the present invention is a bispecific tetravalent antibody, a trispecific hexavalent antibody, or a tetraspecific octavalent antibody; even more preferably, the multispecific antibody, or the antigen binding fragment thereof, according to the present invention is a bispecific tetravalent antibody or a trispecific hexavalent antibody.

In general, it is preferred that the antibody, or the antigen binding fragment thereof, according to the present invention is a monoclonal antibody or antigen binding fragment thereof. Monoclonal antibodies are usually produced by a single clone of B lymphocytes, for example by making hybrid antibody-forming cells, e.g. from a fusion of nonsecreting myeloma cells with immune spleen cells. In contrast to polyclonal antibodies, multispecific monoclonal antibodies bind to (pre)defined epitopes. Therefore, unexpected binding, in particular to undefined epitopes, is largely avoided and monoclonal antibodies are considered as safer compared to polyclonal antibodies.

Preferably, the antibody according to the present invention, or the antigen binding fragment thereof, is a human antibody, a monoclonal antibody, a human monoclonal antibody, or a purified antibody. The antibody according to the present invention, or the antigen binding fragment thereof, may also be a single chain antibody.

Fc Moiety

The multispecific antibody, or antigen binding fragment thereof, according to the present invention comprises an Fc moiety. Preferably, the Fc moiety is derived from human origin, e.g. from human IgG1, IgG2, IgG3, and/or IgG4, whereby human IgG1 is particularly preferred.

As used herein, the term “Fc moiety” refers to a sequence derived from the portion of an immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site (e.g., residue 216 in native IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C-terminus of the immunoglobulin heavy chain. Accordingly, an Fc moiety may be a complete Fc moiety or a portion (e.g., a domain) thereof. A complete Fc moiety comprises at least a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid positions 216-446). An additional lysine residue (K) is sometimes present at the extreme C-terminus of the Fc moiety, but is often cleaved from a mature antibody. Each of the amino acid positions within an Fc region have been numbered according to the art-recognized EU numbering system of Kabat, see e.g., by Kabat et al., in “Sequences of Proteins of Immunological Interest”, U.S. Dept. Health and Human Services, 1983 and 1987.

Preferably, in the context of the present invention an Fc moiety comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant, portion, or fragment thereof. In preferred embodiments, an Fc moiety comprises at least a hinge domain, a CH2 domain or a CH3 domain. More preferably, the Fc moiety is a complete Fc moiety. The Fc moiety may also comprises one or more amino acid insertions, deletions, or substitutions relative to a naturally-occurring Fc moiety. For example, at least one of a hinge domain, CH2 domain or CH3 domain (or portion thereof) may be deleted. For example, an Fc moiety may comprise or consist of: (i) hinge domain (or portion thereof) fused to a CH2 domain (or portion thereof), (ii) a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof), (iii) a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof), (iv) a hinge domain (or portion thereof), (v) a CH2 domain (or portion thereof), or (vi) a CH3 domain or portion thereof.

It will be understood by one of ordinary skill in the art that the Fc moiety may be modified such that it varies in amino acid sequence from the complete Fc moiety of a naturally occurring immunoglobulin molecule, while retaining at least one desirable function conferred by the naturally-occurring Fc moiety. Such functions include Fc receptor (FcR) binding, antibody half-life modulation, ADCC function, protein A binding, protein G binding, and complement binding. The portions of naturally-occurring Fc moieties, which are responsible and/or essential for such functions are well known by those skilled in the art.

For example, to activate the complement cascade C1q binds to at least two molecules of IgG1 or one molecule of IgM, attached to the antigenic target (Ward, E. S., and Ghetie, V., Ther. Immunol. 2 (1995) 77-94). Burton, D. R., described (Mol. Immunol. 22 (1985) 161-206) that the heavy chain region comprising amino acid residues 318 to 337 is involved in complement fixation. Duncan, A. R., and Winter, G. (Nature 332 (1988) 738-740), using site directed mutagenesis, reported that Glu318, Lys320 and Lys322 form the binding site to C1q. The role of Glu318, Lys320 and Lys 322 residues in the binding of C1q was confirmed by the ability of a short synthetic peptide containing these residues to inhibit complement mediated lysis.

For example, FcR binding can be mediated by the interaction of the Fc moiety (of an antibody) with Fc receptors (FcRs), which are specialized cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily, and were shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of erythrocytes and various other cellular targets (e.g. tumor cells) coated with the corresponding antibody, via antibody dependent cell mediated cytotoxicity (ADCC; Van de Winkel, J. G., and Anderson, C. L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin classes; Fc receptors for IgG antibodies are referred to as FcγR, for IgE as FcεR, for IgA as FcαR and so on and neonatal Fc receptors are referred to as FcRn. Fc receptor binding is described for example in Ravetch, J. V., and Kinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76 (1998) 231-248.

Cross-linking of receptors by the Fc domain of native IgG antibodies (FcγR) triggers a wide variety of effector functions including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as immune complex clearance and regulation of antibody production. In humans, three classes of FcγR have been characterized, which are: (i) FcγRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) FcγRII (CD32), which binds complexed IgG with medium to low affinity, is widely expressed, in particular on leukocytes, is known to be a central player in antibody-mediated immunity, and which can be divided into FcγRIIA, FcγRIIB and FcγRIIC, which perform different functions in the immune system, but bind with similar low affinity to the IgG-Fc, and the ectodomains of these receptors are highly homologuous; and (iii) FcγRIII (CD16), which binds IgG with medium to low affinity and exists as two types: FcγRIIIA found on NK cells, macrophages, eosinophils and some monocytes and T cells and mediating ADCC and FcγRIIIB, which is highly expressed on neutrophils. FcγRIIA is found on many cells involved in killing (e.g. macrophages, monocytes, neutrophils) and seems able to activate the killing process. FcγRIIB seems to play a role in inhibitory processes and is found on B-cells, macrophages and on mast cells and eosinophils. On B-cells it seems to function to suppress further immunoglobulin production and isotype switching to say for example the IgE class. On macrophages, FcγRIIB acts to inhibit phagocytosis as mediated through FcγRIIA. On eosinophils and mast cells the b form may help to suppress activation of these cells through IgE binding to its separate receptor.

Regarding FcγRI binding, modification in native IgG of at least one of E233-G236, P238, D265, N297, A327 and P329 reduces binding to FcγRI. IgG2 residues at positions 233-236, substituted into IgG1 and IgG4, reduces binding to FcγRI by 10³-fold and eliminated the human monocyte response to antibody-sensitized red blood cells (Armour, K. L., et al. Eur. J. Immunol. 29 (1999) 2613-2624). Regarding FcγRII binding, reduced binding for FcγRIIA is found e.g. for IgG mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414. Regarding FcγRIII binding, reduced binding to FcγRIIIA is found e.g. for mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376. Mapping of the binding sites on human IgG1 for Fc receptors, the above mentioned mutation sites and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604.

Regarding binding to the crucial FcγRII, two regions of native IgG Fc appear to be critical for interactions of FcγRIIs and IgGs, namely (i) the lower hinge site of IgG Fc, in particular amino acid residues L, L, G, G (234-237, EU numbering), and (ii) the adjacent region of the CH2 domain of IgG Fc, in particular a loop and strands in the upper CH2 domain adjacent to the lower hinge region, e.g. in a region of P331 (Wines, B. D., et al., J. Immunol. 2000; 164: 5313-5318). Moreover, FcγRI appears to bind to the same site on IgG Fc, whereas FcRn and Protein A bind to a different site on IgG Fc, which appears to be at the CH2-CH3 interface (Wines, B. D., et al., J. Immunol. 2000; 164: 5313-5318).

For example, the Fc moiety may comprise or consist of at least the portion of an Fc moiety that is known in the art to be required for FcRn binding or extended half-life. Alternatively or additionally, the Fc moiety of the antibody of the invention comprises at least the portion of known in the art to be required for Protein A binding and/or the Fc moiety of the antibody of the invention comprises at least the portion of an Fc molecule known in the art to be required for protein G binding. Preferably, the retained function is the clearance of cytokine-immune complexes, e.g. GM-CSF-immune complexes, which is assumed to be mediated by FcγR binding. Accordingly, a preferred Fc moiety comprises at least the portion known in the art to be required for FcγR binding. As outlined above, a preferred Fc moiety may thus at least comprise (i) the lower hinge site of native IgG Fc, in particular amino acid residues L, L, G, G (234-237, EU numbering), and (ii) the adjacent region of the CH2 domain of native IgG Fc, in particular a loop and strands in the upper CH2 domain adjacent to the lower hinge region, e.g. in a region of P331, for example a region of at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids in the upper CH2 domain of native IgG Fc around P331, e.g. between amino acids 320 and 340 (EU numbering) of native IgG Fc.

Preferably, the multispecific anti-cytokine, preferably anti-GM-CSF, antibody, or antigen binding fragment thereof, according to the present invention comprises an Fc region. As used herein, the term “Fc region” refers to the portion of an immunoglobulin formed by two or more Fc moieties of antibody heavy chains. For example, the Fc region may be monomeric or “single-chain”Fc region (i.e., a scFc region). Single chain Fc regions are comprised of Fc moieties linked within a single polypeptide chain (e.g., encoded in a single contiguous nucleic acid sequence). Exemplary scFc regions are disclosed in WO 2008/143954 A2. Preferably, the Fc region is a dimeric Fc region. A “dimeric Fc region” or “dcFc” refers to the dimer formed by the Fc moieties of two separate immunoglobulin heavy chains. The dimeric Fc region may be a homodimer of two identical Fc moieties (e.g., an Fc region of a naturally occurring immunoglobulin) or a heterodimer of two non-identical Fc moieties.

The Fc moieties of the Fc region may be of the same or different class and/or subclass. For example, the Fc moieties may be derived from an immunoglobulin (e.g., a human immunoglobulin) of an IgG1, IgG2, IgG3 or IgG4 subclass. Preferably, the Fc moieties of Fc region are of the same class and subclass. However, the Fc region (or one or more Fc moieties of an Fc region) may also be chimeric, whereby a chimeric Fc region may comprise Fc moieties derived from different immunoglobulin classes and/or subclasses. For example, at least two of the Fc moieties of a dimeric or single-chain Fc region may be from different immunoglobulin classes and/or subclasses. Additionally or alternatively, the chimeric Fc regions may comprise one or more chimeric Fc moieties. For example, the chimeric Fc region or moiety may comprise one or more portions derived from an immunoglobulin of a first subclass (e.g., an IgG1, IgG2, or IgG3 subclass) while the remainder of the Fc region or moiety is of a different subclass. For example, an Fc region or moiety of an Fc polypeptide may comprise a CH2 and/or CH3 domain derived from an immunoglobulin of a first subclass (e.g., an IgG1, IgG2 or IgG4 subclass) and a hinge region from an immunoglobulin of a second subclass (e.g., an IgG3 subclass). For example, the Fc region or moiety may comprise a hinge and/or CH2 domain derived from an immunoglobulin of a first subclass (e.g., an IgG4 subclass) and a CH3 domain from an immunoglobulin of a second subclass (e.g., an IgG1, IgG2, or IgG3 subclass). For example, the chimeric Fc region may comprise an Fc moiety (e.g., a complete Fc moiety) from an immunoglobulin for a first subclass (e.g., an IgG4 subclass) and an Fc moiety from an immunoglobulin of a second subclass (e.g., an IgG1, IgG2 or IgG3 subclass). For example, the Fc region or moiety may comprise a CH2 domain from an IgG4 immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. For example, the Fc region or moiety may comprise a CH1 domain and a CH2 domain from an IgG4 molecule and a CH3 domain from an IgG1 molecule. For example, the Fc region or moiety may comprise a portion of a CH2 domain from a particular subclass of antibody, e.g., EU positions 292-340 of a CH2 domain. For example, an Fc region or moiety may comprise amino acids a positions 292-340 of CH2 derived from an IgG4 moiety and the remainder of CH2 derived from an IgG1 moiety (alternatively, 292-340 of CH2 may be derived from an IgG1 moiety and the remainder of CH2 derived from an IgG4 moiety).

Moreover, an Fc region or moiety may (additionally or alternatively) for example comprise a chimeric hinge region. For example, the chimeric hinge may be derived, e.g. in part, from an IgG1, IgG2, or IgG4 molecule (e.g., an upper and lower middle hinge sequence) and, in part, from an IgG3 molecule (e.g., an middle hinge sequence). In another example, an Fc region or moiety may comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule. In another example, the chimeric hinge may comprise upper and lower hinge domains from an IgG4 molecule and a middle hinge domain from an IgG1 molecule. Such a chimeric hinge may be made, for example, by introducing a proline substitution (Ser228Pro) at EU position 228 in the middle hinge domain of an IgG4 hinge region. In another embodiment, the chimeric hinge can comprise amino acids at EU positions 233-236 are from an IgG2 antibody and/or the Ser228Pro mutation, wherein the remaining amino acids of the hinge are from an IgG4 antibody (e.g., a chimeric hinge of the sequence ESKYGPPCPPCPAPPVAGP; SEQ ID NO: 191). Further chimeric hinges, which may be used in the Fc moiety of the antibody according to the present invention are described in US 2005/0163783 A1.

Specifically included within the definition of “Fc region” is an “aglycosylated Fc region”. The term “aglycosylated Fc region” refers to an Fc region that lacks a covalently linked oligosaccharide or glycan, e.g., at the N-glycosylation site at EU position 297, in one or more of the Fc moieties thereof. For example, the aglycosylated Fc region may be fully aglycosylated, i.e., all of its Fc moieties lack carbohydrate. Alternatively, the aglycosylated Fc region may be partially aglycosylated (i.e., hemi-glycosylated). The aglycosylated Fc region may be a deglycosylated Fc region, that is an Fc region for which the Fc carbohydrate has been removed, for example chemically or enzymatically. Alternatively or additionally, the aglycosylated Fc region may be a nonglycosylated or unglycosylated, that is an antibody that was expressed without Fc carbohydrate, for example by mutation of one or residues that encode the glycosylation pattern, e.g., at the N-glycosylation site at EU position 297 or 299, by expression in an organism that does not naturally attach carbohydrates to proteins, (e.g., bacteria), or by expression in a host cell or organism whose glycosylation machinery has been rendered deficient by genetic manipulation or by the addition of glycosylation inhibitors (e.g., glycosyltransferase inhibitors). Alternatively, the Fc region is a “glycosylated Fc region”, i.e., it is fully glycosylated at all available glycosylation sites.

In the present invention it is preferred that the Fc moiety, or the Fc region, comprises or consists of an amino acid sequence derived from a human immunoglobulin sequence (e.g., from an Fc region or Fc moiety from a human IgG molecule). However, polypeptides may comprise one or more amino acids from another mammalian species. For example, a primate Fc moiety or a primate binding site may be included in the subject polypeptides. Alternatively, one or more murine amino acids may be present in the Fc moiety or in the Fc region.

Preferably, the multispecific anti-cytokine, preferably anti-GM-CSF, antibody according to the present invention comprises, in particular in addition to an Fc moiety as described above, other parts derived from a constant region, in particular from a constant region of IgG, preferably from a constant region of IgG1, more preferably from a constant region of human IgG1. More preferably, the multispecific antibody according to the present invention comprises, in particular in addition to an Fc moiety as described above, all other parts of the constant regions, in particular all other parts of the constant regions of IgG, preferably all other parts of the constant regions of IgG1, more preferably all other parts of the constant regions of human IgG1.

As outlined above, a particularly preferred multispecific antibody according to the present invention comprises a (complete) Fc region derived from human IgG1. More preferably, the multispecific antibody according to the present invention comprises, in particular in addition to a (complete) Fc region derived from human IgG1 also all other parts of the constant regions of IgG, preferably all other parts of the constant regions of IgG1, more preferably all other parts of the constant regions of human IgG1.

Cytokines

The multispecific anti-cytokine, preferably anti-GM-CSF, antibody, or antigen binding fragment thereof, according to the present invention binds to a cytokine, preferably to GM-CSF. Cytokines are usually small proteins (˜5-20 kDa) that are important in cell signaling. They are released by cells and affect the behavior of other cells, and sometimes affect the behavior of the releasing cell itself. Cytokines include chemokines, interferons, interleukins, lymphokines, tumor necrosis factor, monokines and colony stimulating factors, but generally not hormones. Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells, whereby a given cytokine may be produced by more than one type of cell.

Chemokines mediate chemoattraction (chemotaxis) between cells. Cytokine proteins are classified as chemokines according to behavior and structural characteristics. In addition to being known for mediating chemotaxis, chemokines are all approximately 8-10 kDa in size have four cysteine residues in conserved locations that are key to forming their 3-dimensional shape. These proteins have historically been known under several other names including the SIS family of cytokines, SIG family of cytokines, SCY family of cytokines, Platelet factor-4 superfamily or intercrines. Chemokines can classified into four main subfamilies: CXC, CC, CX3C and XC.

CC chemokine (or β-chemokine) proteins have two adjacent cysteines (amino acids), near their amino terminus. There have been at least 27 distinct members of this subgroup reported for mammals, called CC chemokine ligands (CCL)-1 to -28; CCL10 is the same as CCL9. Chemokines of this subfamily usually contain four cysteines (C4-CC chemokines), but a small number of CC chemokines possess six cysteines (C6-CC chemokines). C6-CC chemokines include CCU, CCL15, CCL21, CCL23 and CCL28. CC chemokines induce the migration of monocytes and other cell types such as NK cells and dendritic cells. Examples of CC chemokine include monocyte chemoattractant protein-1 (MCP-1 or CCL2) which induces monocytes to leave the bloodstream and enter the surrounding tissue to become tissue macrophages. CCL5 (or RANTES) attracts cells such as T cells, eosinophils and basophils that express the receptor CCR5. Increased CCL11 levels in blood plasma are associated with aging (and reduced neurogenesis) in mice and humans. CC chemokine include for example CCU1-CCL28.

The two N-terminal cysteines of CXC chemokines (or α-chemokines) are separated by one amino acid, represented in this name with an “X”. There have been 17 different CXC chemokines described in mammals, that are subdivided into two categories, those with a specific amino acid sequence (or motif) of glutamic acid-leucine-arginine (or ELR for short) immediately before the first cysteine of the CXC motif (ELR-positive), and those without an ELR motif (ELR-negative). ELR-positive CXC chemokines specifically induce the migration of neutrophils, and interact with chemokine receptors CXCR1 and CXCR2. An example of an ELR-positive CXC chemokine is interleukin-8 (IL-8), which induces neutrophils to leave the bloodstream and enter into the surrounding tissue. Other CXC chemokines that lack the ELR motif, such as CXCL13, tend to be chemoattractant for lymphocytes. CXC chemokines bind to CXC chemokine receptors, of which seven have been discovered to date, designated CXCR1-7. CXC chemokine include for example CXCL1-CXCL17.

The third group of chemokines is known as the C chemokines (or γ chemokines), and is unlike all other chemokines in that it has only two cysteines; one N-terminal cysteine and one cysteine downstream. Two chemokines have been described for this subgroup and are called XCL1 (lymphotactin-α) and XCL2 (lymphotactin-β).

A fourth group has also been discovered and members have three amino acids between the two cysteines and is termed CX₃C chemokine (or d-chemokines). The only CX₃C chemokine discovered to date is called fractalkine (or CX₃CL1). It is both secreted and tethered to the surface of the cell that expresses it, thereby serving as both a chemoattractant and as an adhesion molecule.

Interferons (IFNs) are a group of cytokines made and released for example in response to the presence of pathogens, such as viruses, bacteria, parasites, or tumor cells. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses. More than twenty distinct IFN genes and proteins have been identified in animals, including humans. Human interferons have been classified into three major types: Type I IFN, Type II IFN, and Type III IFN based on the type of receptor through which they signal. IFNs belonging to all three classes are important for fighting viral infections and for the regulation of the immune system.

All type I IFNs bind to a specific cell surface receptor complex known as the IFN-α/β receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains. The type I interferons present in humans are IFN-α, IFN-β, IFN-ε, IFN-κ and IFN-ω. In general, type I interferons are produced when the body recognizes a virus has invaded it. They are produced by fibroblasts and monocytes. However, the production of type I IFN-α can be prohibited by another cytokine known as Interleukin-10. Once activated, type I interferons are able to create molecules which prevent the virus from producing and replicating it's RNA and DNA. Overall, IFN-α is suggested to be used to treat hepatitis B and C infections, while IFN-β is suggested to be used to treat multiple sclerosis.

Interferon type II is also known as immune interferon and is activated in particular by Interleukin-12. Furthermore, type II interferons are released by T helper cells, type 1 specifically. However, they are able to block the proliferation of T helper cells type two. The previous results in an inhibition of Th2 immune response and a further induction of Th1 immune response, which leads to the development of debilitating diseases such as multiple sclerosis. IFN type II binds to IFNGR, which consists of IFNGR1 and IFNGR2 chains. In humans an exemplary IFN type II is IFN-γ.

Interferons type III signal through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12). Although discovered more recently than type I and type II IFNs, recent information demonstrates the importance of Type III IFNs in some types of virus infections.

Interleukins are a group of cytokines that were first seen to be expressed by white blood cells (leukocytes). The function of the immune system depends in a large part on interleukins, and rare deficiencies of a number of them have been described, all featuring autoimmune diseases or immune deficiency. The majority of interleukins are synthesized by helper CD4 T lymphocytes, as well as through monocytes, macrophages, and endothelial cells. They promote the development and differentiation of T and B lymphocytes, and hematopoietic cells. Examples of interleukins includes IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36.

Lymphokines are a subset of cytokines that are produced by a lymphocytes. They are protein mediators typically produced by T cells to direct the immune system response by signalling between its cells. Lymphokines have many roles, including the attraction of other immune cells, including macrophages and other lymphocytes, to an infected site and their subsequent activation to prepare them to mount an immune response. Circulating lymphocytes can detect a very small concentration of lymphokine and then move up the concentration gradient towards where the immune response is required. Lymphokines aid B cells to produce antibodies. Important lymphokines secreted by T helper cells include: IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, and Interferon-gamma.

Tumor necrosis factors (or the TNF family) refer to a group of cytokines that can cause cell death (apoptosis). Nineteen proteins were identified as part of the TNF family on the basis of sequence, functional, and structural similarities, including Tumor Necrosis Factor (TNF) (also known as cachectin or TNF alpha), which is a cytokine that has a wide variety of functions, e.g. it can cause cytolysis of certain tumor cell lines, it is involved in the induction of cachexia, it is a potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion, and it can stimulate cell proliferation and induce cell differentiation under certain conditions; Lymphotoxin-alpha (LT-alpha) and lymphotoxin-beta (LT-beta), two related cytokines produced by lymphocytes that are cytotoxic for a wide range of tumor cells in vitro and in vivo; T cell antigen gp39 (CD40L), a cytokine that seems to be important in B-cell development and activation; CD27L, a cytokine that plays a role in T-cell activation and which induces the proliferation of co-stimulated T cells and enhances the generation of cytolytic T cells; CD30L, a cytokine that induces proliferation of T cells; FASL, a cell surface protein involved in cell death; 4-1BBL, an inducible T cell surface molecule that contributes to T-cell stimulation; OX40L, a cell surface protein that co-stimulates T cell proliferation and cytokine production; and TNF-related apoptosis inducing ligand (TRAIL), a cytokine that induces apoptosis.

All these TNF family members seem to form homotrimeric (or heterotrimeric in the case of LT-alpha/beta) complexes that are recognized by their specific receptors. Strong hydrogen bonds between the monomers stabilize the tertiary structure. One such example is the Asn34-Arg82 hydrogen bond in the M. musculus TNF alpha. The PROSITE pattern for this family is located in a beta-strand in the central section of the protein that is conserved across all members. All members of the TNF family, with the exception of the secreted lymphotoxin and a proliferation-inducing ligand (APRIL), are type II transmembrane proteins that protrude from immune cells. Such membrane-bound TNF ligands frequently signal back to the immune cells when they contact and bind their cognate receptors on other cells. Examples of members of the TNF family include CD40LG (TNFSF5); CD70 (TNFSF7); EDA; FASLG (TNFSF6); LTA (TNFSF1); LTB (TNFSF3); TNF, TNFSF4 (OX40L); TNFSF8 (CD153); TNFSF9; TNFSF10 (TRAIL); TNFSF (RANKL); TNFSF12 (TWEAK); TNFSF13; TNFSF13B; TNFSF14; TNFSF15; TNFSF18.

Monokines are cytokines, which are produced primarily by monocytes and macrophages. Examples of monokines include IL-1, TNF alpha, interferon alpha and beta, and colony stimulating factors.

Colony stimulating factors (CSFs) are secreted glycoproteins that bind to receptor proteins on the surfaces of hemopoietic stem cells, thereby activating intracellular signaling pathways that can cause the cells to proliferate and differentiate into a specific kind of blood cell. Colony-stimulating factors are usually soluble, in contrast to other, membrane-bound substances of the hematopoietic microenvironment. This is sometimes used as the definition of CSFs. They transduce by paracrine, endocrine, or autocrine signaling. Examples of colony stimulating factors include CSF1 (also known as “macrophage colony-stimulating factor”), CSF2 (also known as “granulocyte macrophage colony-stimulating factor”; GM-CSF and sargramostim), CSF3 (also known as “granulocyte colony-stimulating factor”; G-CSF and filgrastim), as well as synthetic CSFs, such as Promegapoietin.

In the context of the present invention it is preferred that the cytokine, to which the multispecific anti-cytokine, preferably anti-GM-CSF, antibody binds, in particular with two different domains binding to two different non-overlapping sites in the cytokine, is a colony stimulating factor or an interferon. Among the colony stimulating factors, naturally occurring CSFs are preferred (in particular CSF1, CSF2 (GM-CSF) and CSF3 (G-CSF), and GM-CSF is more preferred. Among interferons, type I and type II interferons are preferred, type I interferons are more preferred, and interferon beta is even more preferred. Thus, the cytokine is preferably GM-CSF or interferon beta, more preferably the cytokine is GM-CSF.

Linkers

Preferably, the antibody, or the antigen binding fragment thereof, according to the present invention further comprises:

-   -   (c) at least one linker.

In general, the linkage between two components of the multispecific anti-cytokine, preferably anti-GM-CSF, antibody according to the present invention may be directly or indirectly, i.e. two components directly adjoin or they may be linked by an additional component of the complex, e.g. by a linker. In particular, some of the components of the multispecific antibody according to the present invention may be directly linked, whereas others are linked by a linker. Preferably, the multispecific antibody according to the present invention comprises a linker in the heavy chain for example between two VH sequences, two VL sequences, and/or a VH sequence and a VL sequence (a “VH sequence” is derived from a heavy chain of a monospecific antibody and thus referred to as “VH”, even though it may be present in the heavy chain or the light chain of the multispecific antibody according to the present invention and a “VL sequence” is derived from a light chain of a monospecific antibody and thus referred to as “VL”, even though it may be present in the heavy chain or the light chain of the multispecific antibody according to the present invention). Accordingly, in the light chain a linker may be preferably present between two VH sequences, two VL sequences, and/or a VH sequence and a VL sequence. Additionally, it is also preferred if a linker is present in the heavy chain of the multispecific antibody according to the present invention between a constant domain, for example CH3 (e.g. in IgG CH1-CH2-CH3), and a VH/VL sequence.

As used herein, the terms “linked”, “fused”, or “fusion”, are used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art. Preferably, the components of the antibody according to the present invention are linked by covalent linkage or attachment of two or more proteins, polypeptides, or fragments thereof, e.g. via their individual peptide backbones, for example through expression of a single protein molecule encoding those components or peptide fragments. Preferred fusions are in frame, i.e., on the level of the encoding nucleic acid molecule two or more open reading frames (ORFs) are fused to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs. Thus, the resulting recombinant fusion protein is a single polypeptide containing two or more protein segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature). Although the reading frame is thus made continuous throughout the fused genetic segments, the protein segments may be physically or spatially separated by, for example, an (in-frame) peptide linker.

Accordingly, the linker, which links two components of the multispecific antibody may be a peptide linker. Alternatively, the linker may also be non-peptidic, e.g. a cross-linking agent, however, a peptide linker is preferred.

A non-peptidic spacer can include or may be an ester, a thioester, and a di-sulfide. Cross-linking agents for peptide or protein crosslinking include for example (i) amine-to-amine crosslinkers, e.g. homobifunctional amine-specific protein crosslinking reagents based on NHS-ester and imidoester reactive groups for selective conjugation of primary amines; available in short, long, cleavable, irreversible, membrane permeable, and cell surface varieties; (ii) sulfhydryl-to-carbohydrate crosslinkers, e.g. crosslinking reagents based on maleimide and hydrazide reactive groups for conjugation and formation of covalent crosslinks; (iii) sulfhydryl-to-sulfhydryl crosslinkers, e.g. homobifunctional sulfhydryl-specific crosslinking reagents based on maleimide or pyridyldithiol reactive groups for selective covalent conjugation of protein and peptide thiols (reduced cysteines) to form stable thioether bonds; (iv) photoreactive crosslinkers, e.g. aryl azide, diazirine, and other photo-reactive (light-activated) chemical heterobifunctional crosslinking reagents to conjugate proteins, nucleic acids and other molecular structures involved in receptor-ligand interaction complexes via two-step activation; (v) amine-to-sulfhydryl crosslinkers, e.g. heterobifunctional protein crosslinking reagents for conjugation between primary amine (lysine) and sulfhydryl (cysteine) groups of proteins and other molecules; available with different lengths and types of spacer arms; and (vi) amine-to-amine crosslinkers, e.g. carboxyl-to-amine crosslinkers, e.g. Carbodiimide crosslinking reagents, DCC and EDC (EDAC), for conjugating carboxyl groups (glutamate, aspartate, C-termini) to primary amines (lysine, N-termini) and also N-hydroxysuccinimide (NHS) for stable activation of carboxylates for amine-conjugation.

A peptidic linker preferably consists of about 1-30 amino acids, whereby a “short linker” consists preferably of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, more preferably of about 1, 2, 3, 4, 5 or 6 amino acids, even more preferably 4-6 amino acids, particularly preferably 5 amino acids, which may be preferably according to SEQ ID NO:143 or a functional sequence variant thereof. A “long linker”, in contrast, consists preferably of about 10-30 amino acids, more preferably of about 12-25 amino acids, and even more preferably of about 14-20 amino acids. A particularly preferred long linker has 15-17 amino acids, preferably 16 amino acids, more preferably according to SEQ ID NO: 144 or a functional sequence variant thereof. In the multispecific antibody according to the present invention a long linker preferably links a VH sequence and a corresponding VL sequence (“corresponding” refers herein to a VH and VL sequence forming together an epitope binding site), e.g. a VH sequence and a VL sequence derived from the same monospecific antibody. A short linker may preferably link VH/VL sequences with “non-corresponding” VH/VL sequences, e.g. from different monospecific antibodies and/or may preferably link a constant domain, for example CH3 (e.g. in IgG CH1-CH2-CH3), with a VH/VL sequence.

Preferred linkers comprise or consist of an amino acid sequence according to SEQ ID NO: 143 or SEQ ID NO: 144 or a functional sequence variant thereof.

Alternatively, the amino acid sequence of the peptidic linker may be identical to that of the N-terminal or C-terminal flanking region. Alternatively a peptidic linker can consist of non-natural amino acid sequences such as an amino acid sequence resulting from conservative amino acid substitutions of said natural flanking regions In a particular embodiment, the peptidic spacer does not contain any Cys (C) residues. In a preferred embodiment the linker sequence contains at least 20%, more preferably at least 40%, even more preferably at least 50%, and particularly preferably at least 70% Gly (G) or β-alanine residues (A). More preferably, the linker sequence contains at least 20%, more preferably at least 40%, even more preferably at least 50%, and particularly preferably at least 70% Gly (G) residues. Appropriate linker sequences can be easily selected and prepared by a person skilled in the art. They may be composed of D and/or L amino acids.

Antibody Formats and Construct Types

In principle, the multispecific antibody according to the present invention, may be of any antibody format as long as the antibody comprises at least two different domains specifically binding to at least two different, non-overlapping sites in a cytokine (e.g. GM-CSF) and an Fc moiety.

For example, the antibody may be a multispecific antibody fragment with an Fc moiety. Examples, in particular for a bispecific antibody fragment with an Fc moiety, are Tandem scFv-Fc, scFv-Fc, scFv-Fc knobs-into-holes, scFv-Fc-scFv, and scDiabody-Fc, which are shown for example in FIG. 3b of Chan, A. C. and Carter, P. J. (2010) Nat Rev Immu 10: 301-316 and described in said article.

The antibody according to the present invention may be based on any immunoglobulin class (e.g., IgA, IgG, IgM etc.) and subclass (e.g. IgA1, IgA2, IgG1, IgG2, IgG3, IgG4 etc.). Preferably, the multispecific antibody according to the present invention is based on IgG (also referred to as “IgG type”). Within the IgG class, antibodies may be based on the IgG1, IgG2, IgG3 or IgG4 subclass, whereby an antibody based on IgG1 (also referred to as “IgG1 type”) is preferred. Preferably, antibodies of the invention may have a κ or a λ light chain.

IgG-based multispecific antibody formats are well-known to the skilled person and preferred IgG-based antibody formats include for example hybrid hybridoma, knobs-into-holes with a common light chain, various IgG-scFv formats, various scFv-IgG formats, two-in-one IgG, dual (or multiple, respectively, e.g. 3 times, 4 times etc.) V domain IgG, IgG-V, and V-IgG, which are shown in Figure 3c of Chan, A. C. and Carter, P. J. (2010) Nat Rev Immu 10: 301-316 and described in said article, for bispecific IgG-based antibodies, or any combination thereof resulting in a multispecific antibody of the IgG-type. Other preferred IgG-based antibody formats include for example DAF, CrossMab, IgG-dsscFv, DVD, IgG-dsFV, IgG-scFab, scFab-dsscFv and Fv2-Fc, which are shown in Fig. 1A of Weidle U. H. et al. (2013) Cancer Genomics and Proteomics 10: 1-18 and described in said article.

Preferably, the multispecific antibody, or the antigen binding fragment thereof, according to the present invention, is of the IgG type, preferably of the IgG1 type, more preferably comprising a heavy chain constant region of the IgG1 CH1-CH2-CH3 type and a light chain constant region of the IgG CK type, even more preferably comprising a heavy chain constant region of the IgG1 CH1-CH2-CH3 type comprising or consisting of an amino acid sequence according to SEQ ID NO: 140 or functional sequence variants thereof, and a light chain constant region of the IgG CK type comprising or consisting of an amino acid sequence according to SEQ ID NO: 141 or functional sequence variants thereof.

Since IgG has two native epitope binding sites, further epitope binding sites, i.e. “non-native” epitope binding sites, are preferably conjugated to the CH3-domain, preferably to the C-terminus of the CH3-domain, of one or both heavy chains constituting the IgG antibody format. Alternatively, further epitope binding sites, i.e. “non-native” epitope binding sites, are preferably conjugated to one or both of the light chain variable regions, preferably to the N-terminus of one or both of the light chain variable regions, of the native epitope binding sites of the IgG antibody format and/or to one or both of the heavy chain variable regions, preferably to the N-terminus of one or both of the heavy chain variable regions, of the native epitope binding sites of the IgG antibody format. Alternatively, further epitope binding sites, i.e. “non-native” epitope binding sites, are preferably conjugated to the CH3-domain, preferably to the C-terminus of the CH3-domain, of one or both heavy chains constituting the IgG antibody format and to one or both of the light chain variable regions, preferably to the N-terminus of one or both of the light chain variable regions, of the native epitope binding sites of the IgG antibody format. Alternatively, further epitope binding sites, i.e. “non-native” epitope binding sites, are preferably conjugated to the CH3-domain, preferably to the C-terminus of the CH3-domain, of one or both heavy chains constituting the IgG antibody format and to one or both of the heavy chain variable regions, preferably to the N-terminus of one or both of the heavy chain variable regions, of the native epitope binding sites of the IgG antibody format.

At any such attachment site of the IgG antibody format, preferably at the light and/or the heavy chain variable regions of the native epitope binding sites and/or the CH3-domain of the heavy chain, one or more than one, e.g. two, three, four or more, non-native epitope binding site(s) may be attached, whereby the attachment of one or two non-native epitope binding site(s) is preferred and the attachment of one non-native epitope binding site is more preferred.

For example, if the antibody according to the present invention, is a bispecific antibody of the IgG format, one “specificity” is preferably provided by a native epitope binding site and the other “specificity” is preferably provided by a non-native epitope binding site. In particular, the non-native epitope binding site may then be attached to any of the above mentioned attachment sites, preferably the light chain variable region of the native epitope binding site, the heavy chain variable region of the native epitope binding site, or the CH3-domain of the heavy chain of the IgG antibody format.

For example, if the antibody according to the present invention, is a trispecific antibody of the IgG format, one “specificity” (“specificity 1”) is preferably provided by a native epitope binding site and the other two “specificities” (“specificity 2” and “specificity 3”) are preferably provided by a non-native epitope binding site. Thereby, the non-native epitope binding site for “specificity 2” may be preferably attached to any of the above mentioned attachment sites, preferably the light chain variable region of the native epitope binding site, the heavy chain variable region of the native epitope binding site, or the CH3-domain of the heavy chain of the IgG antibody format, while the non-native epitope binding site for “specificity 3” may be preferably attached to any other of the above mentioned attachment sites, i.e. to any attachment site, where no non-native epitope binding site for “specificity 2” is attached. Alternatively, the non-native epitope binding sites for the other two “specificities” (“specificity 2” and “specificity 3”) may be preferably attached to the same attachment site, e.g. the non-native epitope binding sites for the other two “specificities” (“specificity 2” and “specificity 3”) are both attached to the light chain variable region of the native epitope binding site, or the non-native epitope binding sites for the other two “specificities” (“specificity 2” and “specificity 3”) are both attached to the heavy chain variable region of the native epitope binding site, or the non-native epitope binding sites for the other two “specificities” (“specificity 2” and “specificity 3”) are both attached to the CH3-domain of the heavy chain of the IgG antibody format. In this case, i.e. the non-native epitope binding sites for the other two “specificities” (“specificity 2” and “specificity 3”) are both attached to the same attachment site, the non-native epitope binding sites for “specificity 2” and the non-native epitope binding sites for “specificity 3” are preferably arranged consecutively, i.e. only one of the non-native epitope binding sites is directly (or via a linker as described herein) attached to the attachment site, whereas the other of the non-native epitope binding sites is preferably linked to the first of the non-native epitope binding sites.

In particular, the antibody according to the present invention preferably comprises two copies of each of the epitope binding sites, whereby it is preferred that the two copies are attached at corresponding positions of the first and the second heavy chain of the IgG-based antibody, e.g. (i) one copy is conjugated to the heavy chain variable region of the native epitope binding sites of the first heavy chain of the IgG antibody format and the other copy is conjugated to the heavy chain variable region of the native epitope binding sites of the second heavy chain of the IgG antibody format; or (ii) one copy is conjugated to the light chain variable region of the native epitope binding sites of the first light chain of the IgG antibody format and the other copy is conjugated to the light chain variable region of the native epitope binding sites of the second light chain of the IgG antibody format; or (iii) one copy is conjugated to the CH3-domain of the first heavy chain of the IgG antibody format and the other copy is conjugated to the CH3-domain of the first light chain of the IgG antibody format.

Preferred antibody formats for the multispecific antibodies according to the present invention, as well as their construction, are described in US 2009/0155275 A1, which relates specifically to multispecific epitope binding proteins comprising an Fc region of an antibody constant domain. Thus, the antibody formats disclosed in US 2009/0155275 A1 are preferably used for the multispecific antibodies according to the present invention.

Exemplary construct types of the antibody according to the present invention include the construct types “Bs1”, “Bs2”, “Bs3”, “Ts1”, “Ts2” and “Ts3”, which are shown in FIG. 4. Accordingly, the multispecific antibody, or the antigen binding fragment thereof, according to the present invention, is preferably according to a construct type selected from the group comprising Bs1, Bs2, Bs3, Ts1, Ts2 and Ts3. More preferably, the multispecific antibody, or the antigen binding fragment thereof, according to the present invention, is according to the construct type Ts3, preferably the antibody, or the antigen binding fragment thereof, is a trispecific antibody according to the construct type Ts3.

Construct type “Bs1” is a bispecific, tetravalent antibody format based on IgG, preferably based on IgG1. A heavy chain of Bs1 comprises (in this order from N to C terminus):

-   (a) a first variable region, e.g. derived from a heavy or light     chain of a first monospecific antibody; preferably a VH sequence,     i.e. a heavy chain variable region, derived from a first     monospecific antibody; -   (b) a corresponding second variable region forming a first epitope     binding site with the first variable region (a), e.g. the     corresponding heavy or light chain variable region derived from a     first monospecific antibody; preferably a VL sequence, i.e. a light     chain variable region, derived from a first monospecific antibody; -   (c) a third variable region, e.g. derived from a heavy chain of a     second monospecific antibody; preferably a VH sequence, i.e. a heavy     chain variable region, derived from a second monospecific antibody;     and -   (d) an IgG CH1-CH2-CH3.

Preferably, components (a) and (b) are linked by a long linker, components (b) and (c) are linked by a short linker and components (c) and (d) are directly linked. A light chain of Bs1 comprises (in this order from N to C terminus):

-   (e) a fourth variable region, forming a second epitope binding site     with the third variable region (c), e.g. the corresponding light     chain variable region derived from a second monospecific antibody;     preferably a VL sequence, i.e. a light chain variable region,     derived from a second monospecific antibody; and -   (f) an IgG CK or IG CL; preferably an Ig CK.

Preferably, components (e) and (f) are directly linked. Since Bs1 is based on IgG, Bs1 comprises two identical heavy chains and two identical light chains. As used herein, the position in the antibody, where the CDRs/variable regions forming the first epitope binding site in Bs (components (a) and (b)) are arranged, is referred to as “position A” and the position in the antibody, where the CDRs/variable regions forming the second epitope binding site in Bs (components (c) and (d)) are arranged, is referred to as “position B” (cf FIG. 4). In construction, a (second) monospecific antibody, from which heavy and light chain variable regions are used in the heavy and the light chain, respectively, of the multispecific antibody, may serve as “scaffold”. A preferred embodiment of construct type “Bs1” and its construction is described in US 2009/0155275 A1, FIGS. 4E and 4F and the corresponding description.

Construct type “Bs2” is a bispecific, tetravalent antibody format based on IgG, preferably based on IgG1. A heavy chain of Bs2 comprises (in this order from N to C terminus):

-   (a) a first variable region, e.g. derived from a heavy chain of a     first monospecific antibody; preferably a VH sequence, i.e. a heavy     chain variable region, derived from a first monospecific antibody;     and -   (b) an IgG CH1-CH2-CH3.

Preferably, components (a) and (b) are directly linked. A light chain of Bs2 comprises (in this order from N to C terminus):

-   (c) a second variable region, e.g. derived from a heavy or light     chain of a second monospecific antibody; preferably a VH sequence,     i.e. a heavy chain variable region, derived from a second     monospecific antibody; -   (d) a corresponding third variable region forming a first epitope     binding site with the second variable region (c), e.g. the     corresponding heavy or light chain variable region derived from a     second monospecific antibody; preferably a VL sequence, i.e. a light     chain variable region, derived from a second monospecific antibody; -   (e) a fourth variable region, forming a second epitope binding site     with the first variable region (a), e.g. the corresponding light     chain variable region derived from a first monospecific antibody;     preferably a VL sequence, i.e. a light chain variable region,     derived from a first monospecific antibody; and -   (f) an IgG CK or IG CL; preferably an Ig CK.

Preferably, components (c) and (d) are linked by a long linker, components (d) and (e) are linked by a short linker and components (e) and (f) are directly linked. Since Bs2 is based on IgG, Bs2 comprises two identical heavy chains and two identical light chains. As used herein, the position in the antibody, where the CDRs/variable regions forming the first epitope binding site in Bs2 (components (c) and (d)) are arranged, is referred to as “position A” and the position in the antibody, where the CDRs/variable regions forming the second epitope binding site in Bs2 (components (a) and (e)) are arranged, is referred to as “position B” (cf FIG. 4). In construction, a (first) monospecific antibody, from which heavy and light chain variable regions are used in the heavy and the light chain, respectively, of the multispecific antibody, may serve as “scaffold”. A preferred embodiment of construct type “Bs2” and its construction is described in US 2009/0155275 A1, FIGS. 4C and 4D and the corresponding description.

Construct type “Bs3” is a bispecific, tetravalent antibody format based on IgG, preferably based on IgG1. A heavy chain of Bs3 comprises (in this order from N to C terminus):

-   (a) a first variable region, e.g. derived from a heavy chain of a     first monospecific antibody; preferably a VH sequence, i.e. a heavy     chain variable region, derived from a first monospecific antibody; -   (b) an IgG CH1-CH2-CH3; -   (c) a second variable region, e.g. derived from a heavy or light     chain of a second monospecific antibody; preferably a VH sequence,     i.e. a heavy chain variable region, derived from a second     monospecific antibody; and -   (d) a corresponding third variable region forming a first epitope     binding site with the second variable region (c), e.g. the     corresponding heavy or light chain variable region derived from a     second monospecific antibody; preferably a VL sequence, i.e. a light     chain variable region, derived from a second monospecific antibody.

Preferably, components (a) and (b) are directly linked, components (b) and (c) are linked by a short linker and components (c) and (d) are linked by a long linker. A light chain of Bs3 comprises (in this order from N to C terminus):

-   (e) a fourth variable region, forming a second epitope binding site     with the first variable region (a), e.g. the corresponding light     chain variable region derived from a first monospecific antibody;     preferably a VL sequence, i.e. a light chain variable region,     derived from a first monospecific antibody; and -   (f) an IgG CK or IG CL; preferably an Ig CK.

Preferably, components (e) and (f) are directly linked. Since Bs3 is based on IgG, Bs3 comprises two identical heavy chains and two identical light chains. As used herein, the position in the antibody, where the CDRs/variable regions forming the first epitope binding site in Bs3 (components (c) and (d)) are arranged, is referred to as “position C” and the position in the antibody, where the CDRs/variable regions forming the second epitope binding site in Bs3 (components (a) and (e)) are arranged, is referred to as “position B” (cf FIG. 4). In construction, a (first) monospecific antibody, from which heavy and light chain variable regions are used in the heavy and the light chain, respectively, of the multispecific antibody, may serve as “scaffold”. The principle of construct type “Bs3” (but with other “specificities”, i.e. epitope binding sites, which are not according to the present invention) and its construction is also described in Dimasi, N., Gao, C., Fleming, R., Woods, R. M., Yao, X.-T., Shirinian, L., Kiener, P. A., and Wu, H. (2009). The design and characterization of oligospecific antibodies for simultaneous targeting of multiple disease mediators. J Mol Biol 393, 672-692: FIG. 1 (d) (“Bs3Ab”) and the corresponding description.

Construct type “Ts1” is a trispecific, hexavalent antibody format based on IgG, preferably based on IgG1. A heavy chain of Ts1 comprises (in this order from N to C terminus):

-   (a) a first variable region, e.g. derived from a heavy chain of a     first monospecific antibody; preferably a VH sequence, i.e. a heavy     chain variable region, derived from a first monospecific antibody; -   (b) an IgG CH1-CH2-CH3; -   (c) a second variable region, e.g. derived from a heavy or light     chain of a second monospecific antibody; preferably a VH sequence,     i.e. a heavy chain variable region, derived from a second     monospecific antibody; -   (d) a corresponding third variable region forming a first epitope     binding site with the second variable region (c), e.g. the     corresponding heavy or light chain variable region derived from a     second monospecific antibody; preferably a VL sequence, i.e. a light     chain variable region, derived from a second monospecific antibody; -   (e) a fourth variable region, e.g. derived from a heavy or light     chain of a third monospecific antibody; preferably a VH sequence,     i.e. a heavy chain variable region, derived from a third     monospecific antibody; and -   (f) a corresponding fifth variable region forming a second epitope     binding site with the fourth variable region (e), e.g. the     corresponding heavy or light chain variable region derived from a     third monospecific antibody; preferably a VL sequence, i.e. a light     chain variable region, derived from a third monospecific antibody.

Preferably, components (a) and (b) are directly linked, components (b) and (c) and components (d) and (e) are linked by a short linker and components (c) and (d) and components (e) and (f) are linked by a long linker. A light chain of Ts1 comprises (in this order from N to C terminus):

-   (g) a sixth variable region, forming a third epitope binding site     with the first variable region (a), e.g. the corresponding light     chain variable region derived from a first monospecific antibody;     preferably a VL sequence, i.e. a light chain variable region,     derived from a first monospecific antibody; and -   (h) an IgG CK or IG CL; preferably an Ig CK.

Preferably, components (g) and (h) are directly linked. Since Ts1 is based on IgG, Ts1 comprises two identical heavy chains and two identical light chains. As used herein, the position in the antibody, where the CDRs/variable regions forming the first epitope binding site in Ts (components (c) and (d)) are arranged, as well as the position in the antibody, where the CDRs/variable regions forming the second epitope binding site in Ts (components (e) and (f)) are arranged, is referred to as “position C”, and the position in the antibody, where the CDRs/variable regions forming the third epitope binding site in Ts (components (a) and (g)) are arranged, is referred to as “position B” (cf FIG. 4). In construction, a (first) monospecific antibody, from which heavy and light chain variable regions are used in the heavy and the light chain, respectively, of the multispecific antibody, may serve as “scaffold”. A preferred embodiment of construct type “Ts1” and its construction is described in US 2009/0155275 A1, FIGS. 3A and 3B and the corresponding description.

Construct type “Ts2” is a trispecific, hexavalent antibody format based on IgG, preferably based on IgG1. A heavy chain of Ts2 comprises (in this order from N to C terminus):

-   (a) a first variable region, e.g. derived from a heavy or light     chain of a first monospecific antibody; preferably a VH sequence,     i.e. a heavy chain variable region, derived from a first     monospecific antibody; -   (b) a corresponding second variable region forming a first epitope     binding site with the first variable region (a), e.g. the     corresponding heavy or light chain variable region derived from a     first monospecific antibody; preferably a VL sequence, i.e. a light     chain variable region, derived from a first monospecific antibody; -   (c) a third variable region, e.g. derived from a heavy chain of a     second monospecific antibody; preferably a VH sequence, i.e. a heavy     chain variable region, derived from a second monospecific antibody;     and -   (d) an IgG CH1-CH2-CH3.

Preferably, components (a) and (b) are linked by a long linker, components (b) and (c) are linked by a short linker and components (c) and (d) are directly linked. A light chain of Ts2 comprises (in this order from N to C terminus):

-   (e) a fourth variable region, e.g. derived from a heavy or light     chain of a third monospecific antibody; preferably a VH sequence,     i.e. a heavy chain variable region, derived from a third     monospecific antibody; -   (f) a corresponding fifth variable region forming a second epitope     binding site with the fourth variable region (e), e.g. the     corresponding heavy or light chain variable region derived from a     third monospecific antibody; preferably a VL sequence, i.e. a light     chain variable region, derived from a third monospecific antibody; -   (g) a sixth variable region, forming a third epitope binding site     with the third variable region (c), e.g. the corresponding light     chain variable region derived from a second monospecific antibody;     preferably a VL sequence, i.e. a light chain variable region,     derived from a second monospecific antibody; and -   (h) an IgG CK or IG CL; preferably an Ig CK.

Preferably, components (e) and (f) are linked by a long linker, components (f) and (g) are linked by a short linker and components (g) and (h) are directly linked. Since Ts2 is based on IgG, Ts2 comprises two identical heavy chains and two identical light chains. As used herein, the position in the antibody, where the CDRs/variable regions forming the first epitope binding site in Ts2 (components (a) and (b)) are arranged as well as the position in the antibody, where the CDRs/variable regions forming the second epitope binding site in Ts2 (components (e) and (f)) are arranged is referred to as “position A”, and the position in the antibody, where the CDRs/variable regions forming the third epitope binding site in Ts2 (components (c) and (g)) are arranged, is referred to as “position B” (cf FIG. 4). In construction, a (second) monospecific antibody, from which heavy and light chain variable regions are used in the heavy and the light chain, respectively, of the multispecific antibody, may serve as “scaffold”. A preferred embodiment of construct type “Ts2” and its construction is described in US 2009/0155275 A1, FIGS. 4G and 4H and the corresponding description.

Construct type “Ts3” is a trispecific, hexavalent antibody format based on IgG, preferably based on IgG1. A heavy chain of Ts3 comprises (in this order from N to C terminus):

-   (a) a first variable region, e.g. derived from a heavy or light     chain of a first monospecific antibody; preferably a VH sequence,     i.e. a heavy chain variable region, derived from a first     monospecific antibody; -   (b) a corresponding second variable region forming a first epitope     binding site with the first variable region (a), e.g. the     corresponding heavy or light chain variable region derived from a     first monospecific antibody; preferably a VL sequence, i.e. a light     chain variable region, derived from a first monospecific antibody; -   (c) a third variable region, e.g. derived from a heavy chain of a     second monospecific antibody; preferably a VH sequence, i.e. a heavy     chain variable region, derived from a second monospecific antibody; -   (d) an IgG CH1-CH2-CH3; -   (e) a fourth variable region, e.g. derived from a heavy or light     chain of a third monospecific antibody; preferably a VH sequence,     i.e. a heavy chain variable region, derived from a third     monospecific antibody; and -   (f) a corresponding fifth variable region forming a second epitope     binding site with the fourth variable region (e), e.g. the     corresponding heavy or light chain variable region derived from a     third monospecific antibody; preferably a VL sequence, i.e. a light     chain variable region, derived from a third monospecific antibody.

Preferably, components (a) and (b) and components (e) and (f) are linked by a long linker, components (b) and (c) and components (d) and (e) are linked by a short linker and components (c) and (d) are directly linked. A light chain of Ts3 comprises (in this order from N to C terminus):

-   (e) a sixth variable region, forming a third epitope binding site     with the third variable region (c), e.g. the corresponding light     chain variable region derived from a second monospecific antibody;     preferably a VL sequence, i.e. a light chain variable region,     derived from a second monospecific antibody; and -   (f) an IgG CK or IG CL; preferably an Ig CK.

Preferably, components (e) and (f) are directly linked. Since Ts3 is based on IgG, Ts3 comprises two identical heavy chains and two identical light chains. As used herein, the position in the antibody, where the CDRs/variable regions forming the first epitope binding site in Ts3 (components (a) and (b)) are arranged, is referred to as “position A”, the position in the antibody, where the CDRs/variable regions forming the second epitope binding site in Ts3 (components (e) and (f)) are arranged, is referred to as “position C”, and the position in the antibody, where the CDRs/variable regions forming the third epitope binding site in Ts3 (components (c) and (d)) are arranged, is referred to as “position B” (cf FIG. 4). In construction, a (second) monospecific antibody, from which heavy and light chain variable regions are used in the heavy and the light chain, respectively, of the multispecific antibody, may serve as “scaffold”.

For further information about the construction of construct types Bs1, Bs2, Bs3, Ts1, and Ts2 the documents US 2009/0155275 A1 and Dimasi, N., Gao, C., Fleming, R., Woods, R. M., Yao, X.-T., Shirinian, L., Kiener, P. A., and Wu, H. (2009): The design and characterization of oligospecific antibodies for simultaneous targeting of multiple disease mediators; J Mol Biol 393, 672-692 may be used. Principles of the constructions outlined in these documents may be adapted to, and then applied to, the novel construct type Ts3.

Neutralization

Preferably, the multispecific antibody, or the antigen binding fragment thereof, according the present invention neutralizes the target effect of a cytokine, in particular GM-CSF,

-   (i) under stringent conditions with an IC₉₀ of 150 ng/ml or less,     preferably with an IC_(go) of 120 ng/ml or less, more preferably     with an IC₉₀ of 100 ng/ml or less, even more preferably with an IC₉₀     of 50 ng/ml or less, and particularly preferably with an IC₉₀ of 10     ng/ml or less; -   (ii) under less stringent conditions with an IC₉₀ of 20 ng/ml or     less, preferably with an IC₉₀ of 15 ng/ml or less, more preferably     with an IC₉₀ of 10 ng/ml or less, even more preferably with an IC₉₀     of 5 ng/ml or less, and particularly preferably with an IC₉₀ of 1     ng/ml or less; -   (iii) under more stringent conditions with an IC₉₀ of 160 ng/ml or     less, preferably with an IC₉₀ of 130 ng/ml or less, more preferably     with an IC₉₀ of 100 ng/ml or less, even more preferably with an IC₉₀     of 50 ng/ml or less, and particularly preferably with an IC₉₀ of 10     ng/ml or less; and/or -   (iv) under very stringent conditions with an IC₉₀ of 1000 ng/ml or     less, preferably with an IC₉₀ of 500 ng/ml or less, more preferably     with an IC₉₀ of 250 ng/ml or less, even more preferably with an IC₉₀     of 100 ng/ml or less, and particularly preferably with an IC₉₀ of 50     ng/ml or less.

Thereby, an “IC₉₀ of x ng/ml or less” refers to the concentration (x ng/ml or less) of the antibody, or the antigen binding fragment thereof, according to the present invention, which is required for 90% neutralization (IC₉₀) of the target effect of a cytokine (e.g. GM-CSF).

In general, the functionality of an antibody is assessed by its ability to neutralize an important effect (“target effect”) of a cytokine (e.g. GM-CSF). In a neutralization assay, the concentration of an antibody required for neutralization, e.g. of a target effect of a cytokine (e.g. GM-CSF) or of infectivity of a virus, can be determined. Various neutralization assays are known to the person skilled in the art, whereby the skilled person typically selects a neutralization assay depending on the cytokine (e.g. GM-CSF) and its target effect. Cytokine (e.g. GM-CSF) target effects which are useful in a neutralization assay include for example cytokine-induced proliferation, e.g. of indicator cell lines, cytokine-induced cytokine production, TNF-α-induced killing of L929 cell line, and IFN-γ-protection from viral infection of L929 and A549 cell lines.

In the following a non-limiting example of a neutralization assay is given to illustrate the principle of a neutralization assay:

-   (1) different concentrations of the antibody to be tested, e.g.     serial dilutions, are prepared for example in a microtiter plate or     any other suitable format; -   (2) a target dose of a cytokine (i.e. a given amount of cytokine,     e.g. GM-CSF) is added to the antibody and co-incubated under     appropriate conditions, e.g. for 1 hour at room temperature or at     37° C.; -   (3) the co-incubated antibody-cytokine (e.g. the co-incubated     antibody-GM-CSF) is transferred to an appropriate target cell     culture, e.g. in wells containing (e.g. monolayers of) target cells,     and incubation is allowed, e.g. at room temperature or 37° C. for a     predetermined amount of time, e.g. 1, 2, 3, 4, 5, 6, or 7 days; and -   (4) thereafter the target effect is analyzed and the IC₉₀ can be     determined.

The effects measured are usually dose-dependent: The higher the antibody titer, the stronger the neutralization of the target effect. Depending on the neutralizing character of the antibody, the IC₉₀ values vary, e.g. an antibody of significant neutralizing character will require lower amounts (of the antibody) to be added for, e.g., achieving the same amount of neutralization of the target effect in the assay.

As used herein, “less stringent conditions” refer to a final cytokine (e.g. GM-CSF) concentration of about 50 pg/ml and about 1000 cells/well. “Stringent conditions” refer to a final cytokine (e.g. GM-CSF) concentration of about 50 pg/ml and about 10000 cells/well. “More stringent conditions” refer to a final cytokine (e.g. GM-CSF) concentration of about 500 pg/ml and about 1000 cells/well. “Very stringent conditions” refer to a final cytokine (e.g. GM-CSF) concentration of about 500 pg/ml and about 10000 cells/well.

Examples of suitable indicator cell lines for assessing neutralization of cytokine-induced proliferation or neutralization of cytokine-induced cytokine production include TF-1, MC/9, L929, D10, CTLL-2, B9, splenocytes, NIH/3T3, COLO205, A549, hPBMC, NHDF and Nag 7/8, whereby TF-1 cells are preferably used in neutralization of the cytokines GM-CSF, IL-13, IL-4, and IL-5, MC/9 cells are preferably used in neutralization of the cytokines GM-CSF, IL-5, and IL-10, L929 cells are preferably used in neutralization of the cytokines IFN-gamma and TNF-alpha, D10 cells are preferably used in neutralization of the cytokines IL-1alpha and IL-1beta, CTLL-2 cells are preferably used in neutralization of the cytokines 11-2, IL-4, and IL-15, B9 cells are preferably used in neutralization of the cytokines IL-6 and IL-21, splenocytes are preferably used in neutralization of the cytokines IL-12/IL-23 p40 and IL-23, NIH/3T3 cells are preferably used in neutralization of the cytokine IL-17A, COLO205 cells are preferably used in neutralization of the cytokine IL-22, A549 cells are preferably used in neutralization of the cytokines IFN-gamma and IFN-beta, hPBMC are preferably used in neutralization of the cytokine IL-12/IL-23 p40, NHDF cells are preferably used in neutralization of the cytokine IL-17A, and Nag7/8 cells are preferably used in neutralization of the cytokine TSLP.

If the ability of an antibody to neutralize the cytokine GM-CSF, in particular GM-CSF-induced proliferation, is assessed, the preferred cells to be used are TF-1 cells. A preferred GM-CSF neutralization assay comprises the following steps:

-   (1) different concentrations of the antibody to be tested, e.g.     serial dilutions, are prepared for example in a microtiter plate or     any other suitable format; -   (2) a target dose of a GM-CSF, e.g. 100 pg/ml, is added to the     antibody and co-incubated under appropriate conditions, in     particular for 1 hour at 37° C.; -   (3) the co-incubated antibody-GM-CSF is transferred to TF-1 cells,     in particular to wells containing 1000 or 10000 TF-1 cells/well, and     incubation is allowed, e.g. at 37° C. for 3-4 days, e.g. 72 h; and -   (4) thereafter the neutralization of proliferation is analyzed and     the IC₉₀ can be determined, for example GM-CSF neutralization may be     calculated as percentage of inhibition of TF-1 growth with the     following formula: [1−(CCPM of a single well−average CCPM of control     cells grown without GM-CSF)/(average CCPM of control cells grown     with GM-CSF−average CCPM of control cells grown without GM-CSF)]×100     (CCPM=corrected counts per minute) and IC₉₀ (□g/ml) may be     calculated for every sample by a nonlinear regression analysis, e.g.     using GraphPad Prism 5 software.

In this assay, less stringent conditions refer to a final GM-CSF concentration of about 50 pg/ml and about 1000 TF-1 cells/well. Stringent conditions refer to a final GM-CSF concentration of about 50 pg/ml and about 10000 TF-1 cells/well. More stringent conditions refer to a final GM-CSF concentration of about 500 pg/ml and about 1000 TF-1 cells/well. Very stringent conditions refer to a final GM-CSF concentration of about 500 pg/ml and about 10000 TF-1 cells/well.

Variable Regions and CDRs

Preferably, the antibody according to the present invention, or the antigen binding fragment thereof, comprises one or more complementarity determining regions (CDRs). In general, complementarity determining regions (CDRs) are the hypervariable regions present in heavy chain variable domains and light chain variable domains of an antibody. Preferably, the antibody according to the present invention, or the antigen binding fragment thereof, comprises at least three CDRs on the heavy chain and at least three CDRs on the light chain. More preferably, the antibody according to the present invention, or the antigen binding fragment thereof, comprises at least six CDRs on the heavy chain and at least three CDRs on the light chain. Even more preferably, the antibody according to the present invention, or the antigen binding fragment thereof, comprises either at least nine CDRs on the heavy chain and at least three CDRs on the light chain or at least six CDRs on the heavy chain and at least six CDRs on the light chain.

Typically, the domain of an antibody, which specifically binds to an epitope of an antigen—e.g. to a certain site in a cytokine molecule, in particular GM-CSF, is also referred to as “antigen receptor” or “epitope binding site”. This domain of the antibody, i.e. the antigen receptor/epitope binding site, is usually, in particular in native monospecific IgG antibodies, formed by the three CDRs of a heavy chain and the three CDRs of the connected light chain. In other words, since in particular in native monospecific IgG antibodies antigen receptors/epitope binding sites are typically composed of two variable domains, there are six CDRs for each antigen receptor (heavy chain: CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3). A single antibody, in particular a single native monospecific IgG antibody, usually has two (identical) antigen receptors and therefore contains twelve CDRs (i.e. 2×six CDRs).

However, the multispecific antibody, or antigen binding fragment thereof, according to the present invention comprises at least two different domains specifically binding to at least two different non-overlapping sites of a cytokine, in particular GM-CSF. Preferably, a single molecule of the multispecific antibody, or antigen binding fragment thereof, according to the present invention comprises two identical domains of each different domain specifically binding to at least two different non-overlapping sites of a cytokine. It is also preferred that a single molecule of the multispecific antibody, or antigen binding fragment thereof, according to the present invention comprises two heavy chains and two light chains. More preferably, a single molecule of the multispecific antibody, or antigen binding fragment thereof, according to the present invention comprises two heavy chains and two light chains whereby the two heavy chains share at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, and particularly preferably 100% sequence identity and/or the two light chains share at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, and particularly preferably 100% sequence identity. Thus, if a single molecule of the multispecific antibody, or antigen binding fragment thereof, according to the present invention comprises two identical domains of each different domain specifically binding to at least two different non-overlapping sites of a cytokine (e.g. GM-CSF), one of said identical domains is preferably comprised by the first heavy and light chains and the other is preferably comprised by the second heavy and light chains of the multispecific antibody.

Due to their “multispecificity”, i.e. the different epitope binding sites, the heavy chain and/or the light chain of the multispecific antibodies, or antigen binding fragments thereof, according to the present invention may (each) comprise more than three CDRs, in particular more than three different CDRs. For example, the multispecific antibody, or antigen binding fragments thereof, according to the present invention may comprise at least two different domains specifically binding to at least two different non-overlapping sites of a cytokine, in particular GM-CSF, wherein each of said at least two different domains is derived from a different monospecific antibody, e.g. of the IgG-type. Since such a monospecific antibody typically comprises three CDRs in the heavy chain and three CDRs in the light chain forming the antigen receptor/epitope binding site, a multispecific antibody according to the present invention may in particular comprise three CDRs of a heavy chain of a first antibody and three CDRs of a light chain of a first antibody, three CDRs of a heavy chain of a second antibody and three CDRs of a light chain of a second antibody, optionally three CDRs of a heavy chain of a third antibody and three CDRs of a light chain of a third antibody etc. Thus, the number of CDRs comprised by a heavy chain and/or a light chain of a multispecific antibody according to the present invention is preferably a multiple of three, for example three, six, nine, twelve, etc. It is thereby also preferred that the sum of the CDRs comprised by both, heavy chain and light chain of a multispecific antibody according to the present invention is a multiple of six, for example six, twelve, eighteen etc.

In particular, in the multispecific antibody, or antigen binding fragment thereof, according to the present invention the heavy chain may also comprise CDRs or variable regions derived from a light chain of a monospecific antibody. For example, in a multispecific antibody according to the present invention the heavy chain may comprise the heavy chain variable region (VH) and the light chain variable region (VL) derived from a first monospecific antibody and the heavy chain variable region (VH) derived from a second monospecific antibody different from the first monospecific antibody, whereas the light chain variable region (VL) derived from the second monospecific antibody is comprised by the light chain of the multispecific antibody according to the present invention. In such a multispecific antibody, the second monospecific antibody may be used in particular as “scaffold”. Accordingly, in a multispecific antibody according to the present invention the heavy chain may comprise one or more, preferably all three, heavy chain CDRs and one or more, preferably all three, light chain CDRs derived from a first monospecific antibody and one or more, preferably all three, heavy chain CDRs derived from a second monospecific antibody different from the first monospecific antibody, whereas one or more, preferably all three, light chain CDRs derived from the second monospecific antibody is comprised by the light chain of the multispecific antibody according to the present invention. In such multispecific antibodies, the second monospecific antibody may be used in particular as “scaffold”.

Typically, in particular in native monospecific IgG antibodies, the three CDRs (CDR1, CDR2, and CDR3) are arranged non-consecutively in the variable domain. In other words, the CDRs on the heavy and/or light chain may be separated for example by framework regions, whereby a framework region (FR) is a region in the variable domain which is less “variable” than the CDR. For example, in an antibody according to the present invention a variable region (or each variable region, respectively) may preferably comprise four framework regions, separated by three CDRs.

As described above, in the multispecific antibodies according to the present invention, a single chain, preferably a heavy chain, may comprise more than three CDRs (CDR1, CDR2, and CDR3) and/or more than one variable region as described above. Since an “antigen receptor” is typically characterized by the CDRs, i.e. CDRH1, CDRH2, and CDRH3 as well as CDRL1, CDRL2, and CDRL3, it is preferred in the multispecific antibodies according to the present invention that the CDRs are arranged such, that the order (e.g. CDRH1, CDRH2, and CDRH3 and/or CDRL1, CDRL2, and CDRL3 derived from the same monospecific antibody) is maintained to preserve the “antigen receptor”, i.e. to preserve to ability to specifically bind to a certain site in the antigen (e.g. the cytokine, in particular GM-CSF). This means that for example the order of CDRH1, CDRH2, and CDRH3 derived from a first monospecific antibody in an amino acid stretch is preferably not interrupted by any CDR derived from a second monospecific antibody. Moreover, if a single chain, preferably a heavy chain, of a multispecific antibody according to the present invention comprises CDRs derived from a heavy chain and from a light chain of a first monospecific antibody, the heavy chain CDR(s) are preferably arranged next to the light chain CDR(s) derived from the same monospecific antibody. For example, such an arrangement may be -CDRH1(a)-CDRH2(a)-CDRH3(a)-CDRL1(a)-CDRL2(a)-CDRL3(a)-CDRH1(b)-CDRH2(b)-CDRH3(b)-, whereby (a) and (b) refers to different monospecific antibodies from which the respective CDR is derived and the CDRs are usually arranged in a non-consecutive manner, i.e. the CDRs may be separated by any amino acid sequence which is not a CDR, e.g. a framework region and/or a linker. Importantly, if the multipecific antibody according to the present invention comprises epitope binding sites (antigen receptors) derived from at least two different monospecific antibodies, the CDRs or variable regions of these monospecific antibodies are arranged in the multipecific antibody according to the present invention such that the “antigen receptor” of each monospecific antibody from which the CDRs (or variable regions) are derived, is preserved, i.e. its ability to specifically bind to a certain site in the antigen (e.g. the cytokine, in particular GM-CSF), is preserved.

The position of the CDR amino acids are defined herein according to the IMGT numbering system (IMGT: http://www.imgt.org/; cf Lefranc, M.-P. et al. (2009) Nucleic Acids Res. 37, D1006-D1012). Example sequences of CDRs, heavy chains, light chains as well as the sequences of the nucleic acid molecules encoding the CDRs, heavy chains, light chains of the antibodies of the invention, i.e. of several antibodies according to the invention, are disclosed in the sequence listing. The CDRs of a multispecific antibody according to the present invention, which are derived from a heavy chain CDR of a monospecific antibody are also referred to as CDRH1, CDRH2 and CDRH3, respectively. Similarly, the CDRs of a multispecific antibody according to the present invention, which are derived from a light chain CDR of a monospecific antibody are also referred to as CDRL1, CDRL2 and CDRL3, respectively. Thus, for example CDRL1, CDRL2 and CDRL3 may also be present on a heavy chain of a multispecific antibody according to the present invention. Accordingly, the variable regions of a multispecific antibody according to the present invention, which are derived from a heavy chain variable region of a monospecific antibody are also referred to as VH and the variable regions of a multispecific antibody according to the present invention, which are derived from a light chain variable region of a monospecific antibody are also referred to as VL. Thus, for example VL may also be present on a heavy chain of a multispecific antibody according to the present invention.

Preferably, the antibody according to the present invention, or the antigen binding fragment thereof, comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein the at least one heavy chain CDRH3 comprises an amino acid sequence according to SEQ ID NOs: 3, 51, 69, or 107 or functional sequence variants thereof, preferably the at least one heavy chain CDRH3 comprises an amino acid sequence according to SEQ ID NOs: 3 or 69 or functional sequence variants thereof. It is also preferred that the heavy chain comprises at least two CDRH1, at least two CDRH2 and at least two CDRH3, wherein one CDRH1, CDRH2 and CDRH3 is derived from a first monospecific antibody and one CDRH1, CDRH2 and CDRH3 is derived from a second monospecific antibody different from the first monospecific antibody.

It is also preferred that, the multispecific antibody of the invention, or the antigen binding fragment thereof, comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein the at least one heavy chain CDRH3 comprises an amino acid sequence that is at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 3, 51, 69, or 107, preferably to SEQ ID NOs: 3 or 69.

Table 1 provides the SEQ ID numbers for the amino acid sequences of the six CDRs, which are derived from exemplary monospecific antibodies and used in the exemplary multispecific antibodies of the invention.

TABLE 1 SEQ ID Numbers for CDR peptides derived from exemplary monospecific antibodies. Origin (mono- SEQ ID NOs. for CDR peptides Specific AB) CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 GCA7 1 2 3 4 5/6 7 GCA21 49 50 51 52 53/54 55 GCB59 67 68 69 70 71/72 73 GCE536 105 106 107 108 109/110 111

Variant antibodies are also included within the scope of the invention. Thus, variants of the sequences recited in the application are also included within the scope of the invention. Such variants include natural variants generated by somatic mutation in vivo during the immune response or in vitro upon culture of immortalized B cell clones. Alternatively, variants may arise due to the degeneracy of the genetic code or may be produced due to errors in transcription or translation.

Further variants of the antibody sequences having improved affinity and/or potency may be obtained using methods known in the art and are included within the scope of the invention. For example, amino acid substitutions may be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence may be used to improve the efficiency of translation in expression systems for the production of the antibody. Further, polynucleotides comprising a sequence optimized for antibody specificity or neutralizing activity by the application of a directed evolution method to any of the nucleic acid sequences of the invention are also within the scope of the invention.

Preferably, variant antibody sequences may share 70% or more (i.e. 75%, 80%, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity with the sequences recited in the application. Such variants usually have a greater homology to the sequences listed herein in the CDRs of the heavy chain variable region (V_(H)) and light chain variable region (V_(L)) than in the framework region. As is known to one of skill in the art, mutations are more tolerated, i.e., limited or no loss of function (e.g., specificity or neutralization ability) in the framework regions than in the CDRs.

The invention thus comprises an antibody, or an antigen binding fragment thereof, wherein the variation from the sequences provided herein is preferably in the framework region(s) of the antibody or in the nucleic acid residues that encode the framework region(s) of the antibody.

In the present invention, such (variant) antibodies are preferred, in which the number of somatic mutations is reduced (i.e. “germlined” antibodies: reverted back to the “germline” configuration). Germline sequences of antibodies may be determined, for example, with reference to IMGT database (e.g., according to the IMGT VDJ and VJ assignments and rearrangement interpretation: http://www.imgt.org/; cf Lefranc, M.-P. et al. (2009) Nucleic Acids Res. 37, D1006-D1012) and “germlined” antibody variants may be produced, for example, by gene synthesis or by site-directed mutagenesis. A low level of somatic mutations reduces the potential risk of antibody immunogenicity. Preferably, the number of somatic mutations is reduced in the framework regions (FR) (i.e. “framework regions germlined” antibodies, also referred to herein as FR-GL variants). (Variant) antibodies, or an antigen binding fragment thereof, and FR-GL variants, respectively, without any somatic mutations in the framework regions (FR) are more preferred. Particularly preferred are such (variant) antibodies, or an antigen binding fragment thereof, and FR-GL variants, respectively, with as few somatic mutations as possible, whereby on the other hand the neutralizing activity is not impaired (as compared to the reference antibody/fragment containing (more) somatic mutations). Such antibodies are on the one hand not impaired in their neutralizing activities, thus showing a very high potency and breadth. On the other hand, a potential risk of antibody immunogenicity is significantly reduced.

In a preferred embodiment, the multispecific antibody or antibody fragment of the invention comprises at least one CDR with a sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111.

Preferably, the multispecific antibody or antibody fragment of the invention comprises more than one CDR with a sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111.

Preferably, the antibody, or antigen binding fragment thereof, comprises two CDRs with a sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111. Thereby it is preferred that the antibody, or antigen binding fragment thereof, comprises (i) a CDRH1 that has at least 95% sequence identity to any one of SEQ ID NOs: 1, 49, 67 and 105 and a CDRL1 that has at least 95% sequence identity to any one of SEQ ID NOs: 4, 52, 70 and 108; (ii) a CDRH2 that has at least 95% sequence identity to any one of SEQ ID NOs: 2, 50, 68 and 106, and a CDRL2 that has at least 95% sequence identity to any one of SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109 and 110; or (iii) a CDRH3 that has at least 95% sequence identity to any one of SEQ ID NOs: 3, 51, 69 and 107, and a CDRL3 that has at least 95% sequence identity to any one of SEQ ID NOs: 7, 55, 73 and 111.

Preferably, the antibody, or antigen binding fragment thereof, comprises three CDRs with a sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111. Thereby it is preferred that the antibody, or antigen binding fragment thereof, comprises (i) a CDRH1 that has at least 95% sequence identity to any one of SEQ ID NOs: 1, 49, 67 and 105, a CDRH2 that has at least 95% sequence identity to any one of SEQ ID NOs: 2, 50, 68 and 106, and a CDRH3 that has at least 95% sequence identity to any one of SEQ ID NOs: 3, 51, 69 and 107; or (ii) a CDRL1 that has at least 95% sequence identity to any one of SEQ ID NOs: 4, 52, 70 and 108, a CDRL2 that has at least 95% sequence identity to any one of SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109 and 110, and a CDRL3 that has at least 95% sequence identity to any one of SEQ ID NOs: 7, 55, 73 and 111.

Preferably, the antibody, or antigen binding fragment thereof, comprises four CDRs with a sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111. Thereby it is preferred that the antibody, or antigen binding fragment thereof, comprises (i) a CDRH1 that has at least 95% sequence identity to any one of SEQ ID NOs: 1, 49, 67 and 105, a CDRH2 that has at least 95% sequence identity to any one of SEQ ID NOs: 2, 50, 68 and 106, a CDRH3 that has at least 95% sequence identity to any one of SEQ ID NOs: 3, 51, 69 and 107, and a CDRL that has at least 95% sequence identity to any one of SEQ ID NOs: 4-7, 52-55, 70-73, or 108-111; (ii) a CDRL1 that has at least 95% sequence identity to any one of SEQ ID NOs: 4, 52, 70 and 108, a CDRL2 that has at least 95% sequence identity to any one of SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109 and 110, a CDRL3 that has at least 95% sequence identity to any one of SEQ ID NOs: 7, 55, 73 and 111, and a CDRH that has at least 95% sequence identity to any one of SEQ ID NOs: 1-3, 49-51, 67-69, and 105-107, whereby a CDRH3 that has at least 95% sequence identity to any one of SEQ ID NOs: 3, 51, 69 and 107 is particularly preferred; (iii) a CDRH1 that has at least 95% sequence identity to any one of SEQ ID NOs: 1, 49, 67 and 105, a CDRL1 that has at least 95% sequence identity to any one of SEQ ID NOs: 4, 52, 70 and 108, a CDRH2 that has at least 95% sequence identity to any one of SEQ ID NOs: 2, 50, 68 and 106, and a CDRL2 that has at least 95% sequence identity to any one of SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109 and 110; (iv) a CDRH1 that has at least 95% sequence identity to any one of SEQ ID NOs: 1, 49, 67 and 105, a CDRL1 that has at least 95% sequence identity to any one of SEQ ID NOs: 4, 52, 70 and 108, a CDRH3 that has at least 95% sequence identity to any one of SEQ ID NOs: 3, 51, 69 and 107, and a CDRL3 that has at least 95% sequence identity to any one of SEQ ID NOs: 7, 55, 73 and 111; or (v) a CDRH2 that has at least 95% sequence identity to any one of SEQ ID NOs: 2, 50, 68 and 106, a CDRL2 that has at least 95% sequence identity to any one of SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109 and 110, a CDRH3 that has at least 95% sequence identity to any one of SEQ ID NOs: 3, 51, 69 and 107, and a CDRL3 that has at least 95% sequence identity to any one of SEQ ID NOs: 7, 55, 73 and 111.

Preferably, the antibody, or antigen binding fragment thereof, comprises five CDRs with a sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111. Thereby it is preferred that the antibody, or antigen binding fragment thereof, comprises five CDRs selected from the group of a CDRH1 that has at least 95% sequence identity to any one of SEQ ID NOs: 1, 49, 67 and 105, a CDRH2 that has at least 95% sequence identity to any one of SEQ ID NOs: 2, 50, 68 and 106, a CDRH3 that has at least 95% sequence identity to any one of SEQ ID NOs: 3, 51, 69 and 107, a CDRL1 that has at least 95% sequence identity to any one of SEQ ID NOs: 4, 52, 70 and 108, a CDRL2 that has at least 95% sequence identity to any one of SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109 and 110, and a CDRL3 that has at least 95% sequence identity to any one of SEQ ID NOs: 7, 55, 73 and 111.

Preferably, the antibody, or antigen binding fragment thereof, comprises six CDRs with a sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 1-7, 49-55, 67-73 and 105-111. Thereby it is preferred that the antibody, or antigen binding fragment thereof, comprises six CDRs selected from the group of a CDRH1 that has at least 95% sequence identity to any one of SEQ ID NOs: 1, 49, 67 and 105, a CDRH2 that has at least 95% sequence identity to any one of SEQ ID NOs: 2, 50, 68 and 106, a CDRH3 that has at least 95% sequence identity to any one of SEQ ID NOs: 3, 51, 69 and 107, a CDRL1 that has at least 95% sequence identity to any one of SEQ ID NOs: 4, 52, 70 and 108, a CDRL2 that has at least 95% sequence identity to any one of SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109 and 110, and a CDRL3 that has at least 95% sequence identity to any one of SEQ ID NOs: 7, 55, 73 and 111. More preferably, the antibody, or antigen binding fragment thereof, comprises:

-   (i) heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and     light chain CDRL1, CDRL2, and CDRL3 amino acid sequences according     to SEQ ID NOs: 1-5 and 7, or functional sequence variants thereof,     or according to SEQ ID NOs: 1-4 and 6-7, respectively, or functional     sequence variants thereof; -   (ii) heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and     light chain CDRL1, CDRL2, and CDRL3 amino acid sequences according     to SEQ ID NOs: 49-53 and 55, or functional sequence variants     thereof, or according SEQ ID NOs: 49-52 and 54-55, respectively, or     functional sequence variants thereof; -   (iii) heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and     light chain CDRL1, CDRL2, and CDRL3 amino acid sequences according     to SEQ ID NOs: 67-71 and 73, or functional sequence variants     thereof, or according to SEQ ID NOs: 67-70 and 72-73, respectively,     or functional sequence variants thereof; and/or -   (iv) heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and     light chain CDRL1, CDRL2, and CDRL3 amino acid sequences according     to SEQ ID NOs: 105-109 and 111, or functional sequence variants     thereof, or according to SEQ ID NOs: 105-108 and 110-111,     respectively, or functional sequence variants thereof.

Among the embodiments described above of the antibody, or antigen binding fragment thereof, of the invention having at least one CDR, i.e. one, two, three, four, five, six or more CDRs as described above, such an embodiment of the antibody, or antigen binding fragment thereof, is preferred, which comprises a CDRH3 that has at least 95% sequence identity to any one of SEQ ID NOs: 3, 51, 69 and 107.

It is also preferred that, the isolated antibody or antigen binding fragment of the invention comprises a heavy chain CDR1 with the amino acid sequence of SEQ ID NOs: 1, 49, 67 and 105 or functional sequence variants thereof; a heavy chain CDR2 with the amino acid sequence of SEQ ID NOs: 2, 50, 68 and 106 or functional sequence variants thereof; and a heavy chain CDR3 with the amino acid sequence of SEQ ID NOs: 3, 51, 69 and 107 or functional sequence variants thereof. In certain embodiments, an antibody or antibody fragment as provided herein comprises a heavy chain comprising the amino acid sequence of (i) SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2 and SEQ ID NO: 3 for CDRH3, (ii) SEQ ID NO: 49 for CDRH1, SEQ ID NO: 50 for CDRH2, and SEQ ID NO: 21 for CDRH3, (iii) SEQ ID NO: 67 for CDRH1, SEQ ID NO: 68 for CDRH2, and SEQ ID NO: 69 for CDRH3, and/or (iv) SEQ ID NO: 105 for CDRH1, SEQ ID NO: 106 for CDRH2, and SEQ ID NO: 107 for CDRH3.

Preferably, the antibody or antigen binding fragment of the invention comprises a light chain CDR1 with an amino acid sequence according to any of SEQ ID NOs: 4, 52, 70 and 108 or functional sequence variants thereof; a light chain CDR2 with an amino acid sequence according to any of SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109 and 110 or functional sequence variants thereof; and/or a light chain CDR3 with an amino acid sequence according to any of SEQ ID NO: 7, 55, 73 and 111 or functional sequence variants thereof. In certain embodiments, an antibody or antibody fragment as provided herein comprises a light chain comprising the amino acid sequence of (i) SEQ ID NO: 4 for CDRL1, SEQ ID NO: 5 or 6 for CDRL2, and SEQ ID NO: 7 for CDRL3; (ii) SEQ ID NO: 52 for CDRL1, SEQ ID NO: 53 or 54 for CDRL2, and SEQ ID NO: 55 for CDRL3; (iii) SEQ ID NO: 70 for CDRL1, SEQ ID NO: 71 or 72 for CDRL2, and SEQ ID NO: 73 for CDRL3; and/or (iv) SEQ ID NO: 108 for CDRL1, SEQ ID NO: 109 or 110 for CDRL2, and SEQ ID NO: 111 for CDRL3.

In another embodiment of the invention, the invention comprises an isolated antibody or antigen binding fragment thereof, comprising heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2, and CDR3 amino acid sequences that are at least 80%, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any six amino acid sequences of SEQ ID NOs: 1-7, 49-55, 67-73, and 105-111, respectively.

Table 2 provides the SEQ ID numbers for the amino acid sequences, as well as the nucleic acid sequences which encode them, of the heavy and light chain variable regions, which are derived from exemplary monospecific antibodies and used in the exemplary multispecific antibodies of the invention.

TABLE 2 SEQ ID Numbers for VH and VL peptides derived from exemplary monospecific antibodies. Origin (mono- VH amino VL amino VH nucleic VL nucleic Specific AB) acid acid acid acid GCA7 37 38 39-43 44-48 GCA21 63 64 65 66 GCB59 95 96  97-100 101-104 GCE536 130 131 132-135 136-139

These sequences are referred to as “VH sequence” or “VL sequence”, respectively, depending whether they are derived from a heavy chain of a monospecific antibody (e.g. GCA7, GCA21, GCB59, or GCE536) or from a light chain of a monospecific antibody (e.g. GCA7, GCA21, GCB59, or GCE536). Thus, in the multispecific antibody according to the present invention, for example the heavy chain may also (e.g. in addition to one or more VH sequences) comprise one or more VL sequences (which were comprised by the light chain of the monospecific antibody from which they are derived).

Preferably, the isolated antibody or antigen binding fragment according to the present invention comprises a VH sequence having an amino acid sequence that is about 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the sequences according to SEQ ID NOs: 37, 63, 95 and 130. In another embodiment, the antibody or antibody fragment comprises a VL sequence having an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the sequences according to SEQ ID NOs: 38, 64, 96, 131.

Preferably, the antibody or antigen binding fragment according to the present invention comprises:

-   (i) a VH amino acid sequence according to SEQ ID NO: 37 or a     functional sequence variant thereof and a VL amino acid sequence     according to SEQ ID NO: 38 or a functional sequence variant thereof;     and/or -   (ii) a VH amino acid sequence according to SEQ ID NO: 63 or a     functional sequence variant thereof and a VL amino acid sequence     according to SEQ ID NO: 64 or a functional sequence variant thereof;     and/or -   (iii) a VH amino acid sequence according to SEQ ID NO: 95 or a     functional sequence variant thereof and a VL amino acid sequence     according to SEQ ID NO: 96 or a functional sequence variant thereof;     and/or -   (iv) a VH amino acid sequence according to SEQ ID NO: 130 or a     functional sequence variant thereof and a VL amino acid sequence     according to SEQ ID NO: 131 or a functional sequence variant     thereof.

Moreover, the heavy chain of the antibody, or the antigen binding fragment thereof, according to the present invention preferably comprises a VL amino acid sequence selected from the amino acids sequences according to SEQ ID NOs: 38, 64, 96 and 131 or functional sequence variants thereof. More preferably the heavy chain of the antibody or antigen binding fragment comprises a VL amino acid sequence selected from the amino acids sequences according to SEQ ID NOs: 38 and 96 or functional sequence variants thereof and even more preferably the heavy chain of the antibody or antigen binding fragment comprises a VL amino acid sequence according to SEQ ID NO: 96 or functional sequence variants thereof.

In the multispecific antibody, or antigen binding fragment thereof, according to the present invention, it is preferred that

-   (i) the antibody or antigen binding fragment is of a construct type     select from the group consisting of the construct types Bs1, Bs2,     Bs3, Ts1, Ts2 and Ts3; and -   (ii) the antibody or antigen binding fragment comprises a CDRH1     amino acid sequence, a CDRH2 amino acid sequence, a CDRH3 amino acid     sequence, a CDRL1 amino acid sequence, a CDRL2 amino acid sequence     and a CDRL3 amino acid sequence selected from the group consisting     of (a) amino acid sequences according to SEQ ID NOs: 1-5 and 7 or     functional sequence variants thereof; (b) amino acid sequences     according to SEQ ID NOs: 1-4 and 6-7 or functional sequence variants     thereof; (c) amino acid sequences according to SEQ ID NOs: 67-71 and     73 or functional sequence variants thereof; (d) amino acid sequences     according to SEQ ID NOs: 67-70 and 72-73 or functional sequence     variants thereof; (e) amino acid sequences according to SEQ ID NOs:     49-53 and 55 or functional sequence variants thereof; (f) amino acid     sequences according to SEQ ID NOs: 49-52 and 54-55 or functional     sequence variants thereof; (g) amino acid sequences according to SEQ     ID NOs: 105-109 and 111 or functional sequence variants thereof;     and (h) amino acid sequences according to SEQ ID NOs: 105-108 and     110-111 or functional sequence variants thereof.

More preferably, the multispecific antibody, or antigen binding fragment thereof, according to the present invention

-   (i) is of a construct type select from the group consisting of the     construct types Bs1, Bs2,

Bs3, Ts1, Ts2 and Ts3; and

-   (ii) comprises at any of the positions A and/or C a CDRH1 amino acid     sequence, a CDRH2 amino acid sequence, a CDRH3 amino acid sequence,     a CDRL1 amino acid sequence, a CDRL2 amino acid sequence and a CDRL3     amino acid sequence selected from the group consisting of (a) amino     acid sequences according to SEQ ID NOs: 1-5 and 7 or functional     sequence variants thereof; (b) amino acid sequences according to SEQ     ID NOs: 1-4 and 6-7 or functional sequence variants thereof; (c)     amino acid sequences according to SEQ ID NOs: 67-71 and 73 or     functional sequence variants thereof; and (d) amino acid sequences     according to SEQ ID NOs: 67-70 and 72-73 or functional sequence     variants thereof.

Even more preferably, the multispecific antibody, or antigen binding fragment thereof, according to the present invention

-   (i) is of a construct type select from the group consisting of the     construct types Bs1, Bs2,

Bs3, Ts1, Ts2 and Ts3; and

-   (ii) comprises at any of the positions A and/or C a CDRH1 amino acid     sequence, a CDRH2 amino acid sequence, a CDRH3 amino acid sequence,     a CDRL1 amino acid sequence, a CDRL2 amino acid sequence and a CDRL3     amino acid sequence according to SEQ ID NOs: 67-71 and 73 or     functional sequence variants thereof or according to SEQ ID NOs:     67-70 and 72-73 or functional sequence variants thereof.

Particularly preferably, the multispecific antibody, or antigen binding fragment thereof, according to the present invention

-   (i) is of a construct type select from the group consisting of the     construct types Bs1, Bs2, Bs3, Ts1, Ts2 and Ts3; and -   (ii) comprises at position A a CDRH1 amino acid sequence, a CDRH2     amino acid sequence, a CDRH3 amino acid sequence, a CDRL1 amino acid     sequence, a CDRL2 amino acid sequence and a CDRL3 amino acid     sequence according to SEQ ID NOs: 67-71 and 73 or functional     sequence variants thereof or according to SEQ ID NOs: 67-70 and     72-73 or functional sequence variants thereof.

Preferably, the multispecific antibody, or the antigen binding fragment thereof, according to the present invention is according to gTs1GC1, gTs1GC2a, gTs2GC2b, gTs2GC2c, gTs3GC2d, gTs3GC2e, gBs3GC1a, gBs3GC1b, gBs2GC1c, gBs2GC1d, gBs1GC2a, gBs3GC2b, gBs1GC3a, gBs3GC3b, gBs3GC4, and gBs3GC5, preferably it is according to gTs3GC2d or gBs1GC3a. More preferably, the antibody, or the antigen binding fragment thereof, is Ts1GC1, Ts1GC2a, Ts2GC2b, Ts2GC2c, Ts3GC2d, Ts3GC2e, Bs3GC1a, Bs3GC1b, Bs2GC1c, Bs2GC1d, Bs1GC2a, Bs3GC2b, Bs1GC3a, Bs3GC3b, Bs3GC4, and Bs3GC5, preferably Ts3GC2d or Bs1GC3a.

The present inventors have designed and constructed sixteen multispecific antibodies, which are referred to herein as Ts1GC1, Ts1GC2a, Ts2GC2b, Ts2GC2c, Ts3GC2d, Ts3GC2e, Bs3GC1a, Bs3GC1b, Bs2GC1c, Bs2GC1d, Bs1GC2a, Bs3GC2b, Bs1GC3a, Bs3GC3b, Bs3GC4, and Bs3GC5 (cf Example 5; Table 7). Based on the antibodies Ts1GC1, Ts1GC2a, Ts2GC2b, Ts2GC2c, Ts3GC2d, Ts3GC2e, Bs3GC1a, Bs3GC1b, Bs2GC1c, Bs2GC1d, Bs1GC2a, Bs3GC2b, Bs1GC3a, Bs3GC3b, Bs3GC4, and Bs3GC5, in particular on the combination of VH and VL sequences derived from four monospecific antibodies (GCA7, GCA21, GCB59, GCE536), the terms gTs1GC1, gTs1GC2a, gTs2GC2b, gTs2GC2c, gTs3GC2d, gTs3GC2e, gBs3GC1a, gBs3GC1b, gBs2GC1c, gBs2GC1 d, gBs1 GC2a, gBs3GC2b, gBs1 GC3a, gBs3GC3b, gBs3GC4, and gBs3GC5, as used herein, refer to respective “generic” antibodies, or antigen binding fragments thereof, comprising the specific VH and VL amino acid sequences, encoded by the specific nucleotide sequences, as outlined below.

Table 7 (Example 5) shows the SEQ ID NOs for the amino acid sequences of the heavy chains as well as the SEQ ID NOs for the amino acid sequences of light chains of antibodies Ts1GC1, Ts1GC2a, Ts2GC2b, Ts2GC2c, Ts3GC2d, Ts3GC2e, Bs3GC1a, Bs3GC1b, Bs2GC1c, Bs2GC1d, Bs1GC2a, Bs3GC2b, Bs1GC3a, Bs3GC3b, Bs3GC4, and Bs3GC5, respectively (referred to as “complete sequence” in Table 7), as well as the nucleic acid sequences that encode them. The respective sequences are shown in the “Table of Sequences and SEQ ID Numbers” following the “Examples”.

As used herein, “gTs1GC1” refers to a trispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 63, a VH amino acid sequence according to SEQ ID NO: 37,     a VL amino acid sequence according to SEQ ID NO: 38, a VH amino acid     sequence according to SEQ ID NO: 95 and a VL amino acid sequence     according to SEQ ID NO: 96; and -   (ii) a light chain comprising a VL amino acid sequence according to     SEQ ID NO: 64.

Preferably gTs1GC1 is of the IgG1 type, whereby the light chain of gTs1GC1 further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and the heavy chain of gTs1GC1 further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 37 as well as between the VL amino acid sequence according to SEQ ID NO: 38 and the VH amino acid sequence according to SEQ ID NO: 95 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38 as well as between the VH amino acid sequence according to SEQ ID NO: 95 and the VL amino acid sequence according to SEQ ID NO: 96. Preferably, the construct type of gTs1GC1 is Ts1.

As used herein, “gTs1GC2a” refers to a trispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 130, a VH amino acid sequence according to SEQ ID NO: 37,     a VL amino acid sequence according to SEQ ID NO: 38, a VH amino acid     sequence according to SEQ ID NO: 95 and a VL amino acid sequence     according to SEQ ID NO: 96; and -   (ii) a light chain comprising a VL amino acid sequence according to     SEQ ID NO: 131.

Preferably gTs1GC2a is of the IgG1 type, whereby the light chain of gTs1GC2a further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and the heavy chain of gTs1GC2a further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 37 as well as between the VL amino acid sequence according to SEQ ID NO: 38 and the VH amino acid sequence according to SEQ ID NO: 95 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38 as well as between the VH amino acid sequence according to SEQ ID NO: 95 and the VL amino acid sequence according to SEQ ID NO: 96. Preferably, the construct type of gTs1GC2a is Ts1.

As used herein, “gTs2GC2b” refers to a trispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 95, a VL amino acid sequence according to SEQ ID NO: 96,     and a VH amino acid sequence according to SEQ ID NO: 130; and -   (ii) a light chain comprising a VH amino acid sequence according to     SEQ ID NO: 37, a VL amino acid sequence according to SEQ ID NO: 38,     and a VL amino acid sequence according to SEQ ID NO: 131.

Preferably gTs2GC2b is of the IgG1 type, whereby the light chain of gTs2GC2b further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the VL amino acid sequence according to SEQ ID NO: 38 and the VL amino acid sequence according to SEQ ID NO: 131 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38 and the heavy chain of gTs2GC2b further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged the VL amino acid sequence according to SEQ ID NO: 96 and the VH amino acid sequence according to SEQ ID NO: 130 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 95 and the VL amino acid sequence according to SEQ ID NO: 96. Preferably, the construct type of gTs2GC2b is Ts2.

As used herein, “gTs2GC2c” refers to a trispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 95, a VL amino acid sequence according to SEQ ID NO: 96,     and a VH amino acid sequence according to SEQ ID NO: 37; and -   (ii) a light chain comprising a VH amino acid sequence according to     SEQ ID NO: 130, a VL amino acid sequence according to SEQ ID NO:     131, and a VL amino acid sequence according to SEQ ID NO: 38.

Preferably gTs2GC2c is of the IgG1 type, whereby the light chain of gTs2GC2c further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the VL amino acid sequence according to SEQ ID NO: 131 and the VL amino acid sequence according to SEQ ID NO: 38 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 130 and the VL amino acid sequence according to SEQ ID NO: 131 and the heavy chain of gTs2GC2c further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged the VL amino acid sequence according to SEQ ID NO: 96 and the VH amino acid sequence according to SEQ ID NO: 37 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 95 and the VL amino acid sequence according to SEQ ID NO: 96. Preferably, the construct type of gTs2GC2c is Ts2.

As used herein, “gTs3GC2d” refers to a trispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 95, a VL amino acid sequence according to SEQ ID NO: 96,     a VH amino acid sequence according to SEQ ID NO: 130, a VH amino     acid sequence according to SEQ ID NO: 37 and a VL amino acid     sequence according to SEQ ID NO: 38; and -   (ii) a light chain comprising a VL amino acid sequence according to     SEQ ID NO: 131.

Preferably gTs3GC2d is of the IgG1 type, whereby the light chain of gTs3GC2d further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and the heavy chain of gTs3GC2d further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 37 as well as between the VL amino acid sequence according to SEQ ID NO: 96 and the VH amino acid sequence according to SEQ ID NO: 130 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38 as well as between the VH amino acid sequence according to SEQ ID NO: 95 and the VL amino acid sequence according to SEQ ID NO: 96. Preferably, the construct type of gTs3GC2d is Ts3.

As used herein, “gTs3GC2e” refers to a trispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 95, a VL amino acid sequence according to SEQ ID NO: 96,     a VH amino acid sequence according to SEQ ID NO: 37, a VH amino acid     sequence according to SEQ ID NO: 130 and a VL amino acid sequence     according to SEQ ID NO: 131; and -   (ii) a light chain comprising a VL amino acid sequence according to     SEQ ID NO: 38.

Preferably gTs3GC2e is of the IgG1 type, whereby the light chain of gTs3GC2e further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and the heavy chain of gTs3GC2e further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 130 as well as between the VL amino acid sequence according to SEQ ID NO: 96 and the VH amino acid sequence according to SEQ ID NO: 37 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 130 and the VL amino acid sequence according to SEQ ID NO: 131 as well as between the VH amino acid sequence according to SEQ ID NO: 95 and the VL amino acid sequence according to SEQ ID NO: 96. Preferably, the construct type of gTs3GC2e is Ts3.

As used herein, “gBs3GC1a” refers to a bispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 130, a VH amino acid sequence according to SEQ ID NO: 37,     and a VL amino acid sequence according to SEQ ID NO: 38; and -   (ii) a light chain comprising a VL amino acid sequence according to     SEQ ID NO: 131.

Preferably gBs3GC1a is of the IgG1 type, whereby the light chain of gBs3GC1a further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and the heavy chain of gBs3GC1a further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 37 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38. Preferably, the construct type of gBs3GC1a is Bs3.

As used herein, “gBs3GC1b” refers to a bispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 37, a VH amino acid sequence according to SEQ ID NO: 130,     and a VL amino acid sequence according to SEQ ID NO: 131; and -   (ii) a light chain comprising a VL amino acid sequence according to     SEQ ID NO: 38.

Preferably gBs3GC1b is of the IgG1 type, whereby the light chain of gBs3GC1b further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and the heavy chain of gBs3GC1b further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 130 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 130 and the VL amino acid sequence according to SEQ ID NO: 131. Preferably, the construct type of gBs3GC1b is Bs3.

As used herein, “gBs2GC1c” refers to a bispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 130; and -   (ii) a light chain comprising a VH amino acid sequence according to     SEQ ID NO: 37, a VL amino acid sequence according to SEQ ID NO: 38,     and a VL amino acid sequence according to SEQ ID NO: 131.

Preferably gBs2GC1c is of the IgG1 type, whereby the light chain of gBs2GC1c further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the VL amino acid sequence according to SEQ ID NO: 38 and the VL amino acid sequence according to SEQ ID NO: 131 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38, and the heavy chain of gBs2GC1c further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140. Preferably, the construct type of gBs2GC1c is Bs2.

As used herein, “gBs2GC1d” refers to a bispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 37; and -   (ii) a light chain comprising a VH amino acid sequence according to     SEQ ID NO: 130, a VL amino acid sequence according to SEQ ID NO:     131, and a VL amino acid sequence according to SEQ ID NO: 38.

Preferably gBs2GC1d c is of the IgG1 type, whereby the light chain of gBs2GC1d further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the VL amino acid sequence according to SEQ ID NO: 131 and the VL amino acid sequence according to SEQ ID NO: 38 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 130 and the VL amino acid sequence according to SEQ ID NO: 131, and the heavy chain of gBs2GC1d further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140. Preferably, the construct type of gBs2GC1d is Bs2.

As used herein, “gBs1GC2a” refers to a bispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 95, a VL amino acid sequence according to SEQ ID NO: 96,     and a VH amino acid sequence according to SEQ ID NO: 130; and -   (ii) a light chain comprising a VL amino acid sequence according to     SEQ ID NO: 131.

Preferably gBs1GC2a is of the IgG1 type, whereby the light chain of gBs1GC2a further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and the heavy chain of gBs1GC2a further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally, one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the VL amino acid sequence according to SEQ ID NO: 96 and the VH amino acid sequence according to SEQ ID NO: 130 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 95 and the VL amino acid sequence according to SEQ ID NO: 96. Preferably, the construct type of gBs1GC2a is Bs1.

As used herein, “gBs3GC2b” refers to a bispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 95, a VH amino acid sequence according to SEQ ID NO: 130,     and a VL amino acid sequence according to SEQ ID NO: 131; and -   (ii) a light chain comprising a VL amino acid sequence according to     SEQ ID NO: 96.

Preferably gBs3GC2b is of the IgG1 type, whereby the light chain of gBs3GC2b further comprises an IgG1 CL sequence according to SEQ ID NO: 142 and the heavy chain of gBs3GC2b further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 130 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 130 and the VL amino acid sequence according to SEQ ID NO: 131. Preferably, the construct type of gBs3GC2b is Bs3.

As used herein, “gBs1GC3a” refers to a bispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 95, a VL amino acid sequence according to SEQ ID NO: 96,     and a VH amino acid sequence according to SEQ ID NO: 37; and -   (ii) a light chain comprising a VL amino acid sequence according to     SEQ ID NO: 38.

Preferably gBs1GC3a is of the IgG1 type, whereby the light chain of gBs1GC3a further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and the heavy chain of gBs1GC3a further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally, one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the VL amino acid sequence according to SEQ ID NO: 96 and the VH amino acid sequence according to SEQ ID NO: 37 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 95 and the VL amino acid sequence according to SEQ ID NO: 96. Preferably, the construct type of gBs1GC3a is Bs1.

As used herein, “gBs3GC3b” refers to a bispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 95, a VH amino acid sequence according to SEQ ID NO: 37,     and a VL amino acid sequence according to SEQ ID NO: 38; and -   (ii) a light chain comprising a VL amino acid sequence according to     SEQ ID NO: 96.

Preferably gBs3GC3b is of the IgG1 type, whereby the light chain of gBs3GC3b further comprises an IgG1 CL sequence according to SEQ ID NO: 142 and the heavy chain of gBs3GC3b further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 37 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38. Preferably, the construct type of gBs3GC3b is Bs3.

As used herein, “gBs3GC4” refers to a bispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 63, a VH amino acid sequence according to SEQ ID NO: 130,     and a VL amino acid sequence according to SEQ ID NO: 131; and -   (ii) a light chain comprising a VL amino acid sequence according to     SEQ ID NO: 64.

Preferably gBs3GC4 is of the IgG1 type, whereby the light chain of gBs3GC4 further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and the heavy chain of gBs3GC4 further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 130 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 130 and the VL amino acid sequence according to SEQ ID NO: 131. Preferably, the construct type of gBs3GC4 is Bs3.

As used herein, “gBs3GC5” refers to a bispecific antibody, or antigen binding fragment thereof, comprising:

-   (i) a heavy chain comprising a VH amino acid sequence according to     SEQ ID NO: 63, a VH amino acid sequence according to SEQ ID NO: 37,     and a VL amino acid sequence according to SEQ ID NO: 38; and -   (ii) a light chain comprising a VL amino acid sequence according to     SEQ ID NO: 64.

Preferably gBs3GC5 is of the IgG1 type, whereby the light chain of gBs3GC5 further comprises an IgG1 CK sequence according to SEQ ID NO: 141 and the heavy chain of gBs3GC5 further comprises an IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and, optionally one or more linker sequences, e.g. according to SEQ ID NOs: 143 or 144, whereby a short linker may be preferably arranged between the IgG1 CH1-CH2-CH3 sequence according to SEQ ID NO: 140 and the VH amino acid sequence according to SEQ ID NO: 37 and a long linker may be preferably arranged between the VH amino acid sequence according to SEQ ID NO: 37 and the VL amino acid sequence according to SEQ ID NO: 38. Preferably, the construct type of gBs3GC5 is Bs3.

Preferably, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gTs3GC2d or the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gBs1GC3a. Table 3 below shows which VH or VL sequences (SEQ ID numbers of amino acid sequences) comprise which CDRs (SEQ ID numbers of amino acid sequences), as well as the exemplary monospecific antibodies from which they are derived. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gTs1GC1. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gTs1GC2a. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gTs2GC2b. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gTs2GC2c. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gTs2GC2c. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gTs3GC2d. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gTs3GC2d. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gTs3GC2e. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gBs3GC1a. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gBs3GC1b. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gBs2GC1c. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gBs2GC1d. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gBs1GC2a. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gBs3GC2b. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gBs1GC3a. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gBs3GC3b. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gBs3GC4. Alternatively, the isolated multispecific antibody or antigen binding fragment according to the present invention comprises all of the CDRs comprised by all of the respective VH and VL sequences of gBs3GC5.

TABLE 3 SEQ ID numbers of CDR amino acid sequences  comprised by the VH or VL amino acid sequences  as indicated in the table. VL or VH sequence and origin (monospecific AB) CDR1 CDR2 CDR3 GCA7 VH (SEQ ID NO: 37)   1   2   3 GCA7 VL (SEQ ID NO: 38)   4   5, 6   7 GCA21 VH (SEQ ID NO: 63)  49  50  51 GCA21 VL (SEQ ID NO: 64)  52  53, 54  55 GCB59 VH (SEQ ID NO: 95)  67  68  69 GCB59 VL (SEQ ID NO: 96)  70  71, 72  73 GCE536 VH (SEQ ID NO: 130) 105 106 107 GCE536 VL (SEQ ID NO: 131) 108 109, 110 111

The multispecific antibody, or the antigen binding fragment thereof, according to the present invention may be used as a medicament. In particular, the multispecific antibody, or the antigen binding fragment thereof, according to the present invention may be used in the prophylaxis, treatment or attenuation of an inflammatory and/or an autoimmune disease. Further details for this use are described below, e.g. in the context of a pharmaceutical composition and of a medical use.

The invention further comprises an antibody, or fragment thereof, that binds to the same epitopes, i.e. to the same at least two different non-overlapping sites in a cytokine, in particular GM-CSF, as an antibody or antigen binding fragment of the invention, or an antibody that competes with an antibody or antigen binding fragment of the invention.

Preferably, the antibody, or the antigen binding fragment thereof, according to the present invention that binds to the same epitope as the antibody as described above, also comprises an Fc moiety. Preferred embodiments described above for the multispecific antibody, or the antigen binding fragment thereof, according to the present invention that comprises:

-   (a) at least two different domains specifically binding to at least     two different, non-overlapping sites in a cytokine; and -   (b) an Fc moiety     also apply to the antibody, or the antigen binding fragment thereof,     according to the present invention that binds to the same epitope as     the antibody as described above.

Antibodies of the invention also include hybrid multispecific antibody molecules comprising:

-   (a) at least two different domains specifically binding to at least     two different, non-overlapping sites in a cytokine; and -   (b) an Fc moiety     as described above, which comprise one or more CDRs from an antibody     of the invention and one or more CDRs from another antibody, in     particular to the same epitopes or to further epitopes on the same     cytokine, in particular GM-CSF, or on another antigen, e.g. another     cytokine, a tumor associated epitope, a T cell epitope, a B cell     epitope etc. . . . . In one embodiment, such hybrid antibodies     comprise three CDRs from an antibody of the invention and three CDRs     from another antibody, e.g. to the same or other epitopes as     described above. Exemplary hybrid antibodies comprise (i) the three     heavy chain CDRs from an antibody of the invention and the three     light chain CDRs from another antibody to the same or other epitopes     as described above, or (ii) the three light chain CDRs from an     antibody of the invention and the three heavy chain CDRs from     another antibody to the same or other epitopes as described above.

Nucleic Acid Molecule

In another aspect, the invention also provides a nucleic acid molecule comprising a polynucleotide encoding the antibody, or the antigen binding fragment thereof, according to the present invention as described above. Nucleic acid sequences encoding part or all of the light and heavy chains and CDRs of the antibodies of the present invention are preferred. Preferably provided herein are thus nucleic acid sequences encoding part or all of the light and heavy chains, in particular VH and VL sequences and CDRs of the exemplary antibodies of the invention. The SEQ ID numbers for the nucleic acid sequences encoding the VH and VL sequences derived from monospecific antibodies and used in some examples of antibodies of the invention may be derived from Table 7. Table 4 below provides the SEQ ID numbers for the nucleic acid sequences encoding the CDRs of some examples of the antibodies of the invention. Due to the redundancy of the genetic code, the present invention also comprises variants of these nucleic acid sequences encoding the same amino acid sequences.

Thus, the present invention also comprises a nucleic acid molecule comprising a polynucleotide encoding the antibody, or the antigen binding fragment thereof, according to the present invention.

A nucleic acid molecule is a molecule comprising, preferably consisting of nucleic acid components. The term nucleic acid molecule preferably refers to DNA or RNA molecules. In particular, it is used synonymous with the term “polynucleotide”. Preferably, a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers which are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone. The term “nucleic acid molecule” also encompasses modified nucleic acid molecules, such as base-modified, sugar-modified or backbone-modified etc. DNA or RNA molecules.

TABLE 4 SEQ ID Numbers for CDR polynucleotides derived from monospecific antibodies as indicated and used in some exemplary antibodies according to the present invention. Origin (mono- SEQ ID NOs. for CDR polynucleotides Specific AB) CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 GCA7  8-12 13-15 16-20 21-25 26-31 32-36 GCA21 56  57 58 59 60/61 62 GCB59 74-76 77-79 80-82 83-85 86-91 92-94 GCE536 112-114 115 116-118 119-121 122-125 126-129

Preferably, the sequence of the nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence according to any one of SEQ ID NOs: 8-36, 39-48, 56-62, 65-66, 74-94, 97-104, 112-129, 132-139, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, and 190 or a functional sequence variant thereof.

It is also preferred that nucleic acid sequences according to the invention include nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleic acid encoding a VH sequence and/or a VL sequence used in an antibody according to the present invention. Thus a nucleic acid molecule is preferred, wherein the polynucleotide sequence comprises or consists of a nucleic acid sequence according to any one of SEQ ID NOs: 39-48, 65-66, 97-104 and 132-139 or a functional sequence variant thereof. More preferably, a nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence encoding a complete heavy chain or complete light chain of one of the exemplary antibodies according to the present invention. Thus, a nucleic acid molecule is preferred, wherein the polynucleotide sequence comprises or consists of a nucleic acid sequence according to any one of SEQ ID NOs: 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, and 190 or a functional sequence variant thereof.

In another embodiment, a nucleic acid sequence of the invention has the sequence of a nucleic acid encoding a heavy or light chain CDR of an exemplary antibody of the invention. For example, a nucleic acid sequence according to the invention comprises or consists of a sequence that is at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleic acid sequences of SEQ ID NOs: 8-36, 39-48, 56-62, 65-66, 74-94, 97-104, 112-129, 132-139, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, and 190.

In general, the nucleic acid molecule may be manipulated to insert, delete or alter certain nucleic acid sequences. Changes from such manipulation include, but are not limited to, changes to introduce restriction sites, to amend codon usage, to add or optimize transcription and/or translation regulatory sequences, etc. It is also possible to change the nucleic acid to alter the encoded amino acids. For example, it may be useful to introduce one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions and/or insertions into the antibody's amino acid sequence. Such point mutations can modify effector functions, antigen-binding affinity, post-translational modifications, immunogenicity, etc., can introduce amino acids for the attachment of covalent groups (e.g., labels) or can introduce tags (e.g., for purification purposes). Mutations can be introduced in specific sites or can be introduced at random, followed by selection (e.g., molecular evolution). For instance, one or more nucleic acids encoding any of the CDR regions, VH sequence or VL sequence, or a heavy or a light chain of an (exemplary) antibody of the invention can be randomly or directionally mutated to introduce different properties in the encoded amino acids. Such changes can be the result of an iterative process wherein initial changes are retained and new changes at other nucleotide positions are introduced. Further, changes achieved in independent steps may be combined. Different properties introduced into the encoded amino acids may include, but are not limited to, enhanced affinity.

Vector

Further included within the scope of the invention are vectors, for example, expression vectors, comprising a nucleic acid sequence according to the invention. Preferably, a vector comprises a nucleic acid molecule according to the invention, for example a nucleic acid molecule as described above.

The term “vector” refers to a nucleic acid molecule, preferably to an artificial nucleic acid molecule, i.e. a nucleic acid molecule which does not occur in nature. A vector in the context of the present invention is suitable for incorporating or harboring a desired nucleic acid sequence. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc. A storage vector is a vector which allows the convenient storage of a nucleic acid molecule. Thus, the vector may comprise a sequence corresponding, e.g., to a desired antibody or antibody fragment thereof according to the present invention. An expression vector may be used for production of expression products such as RNA, e.g. mRNA, or peptides, polypeptides or proteins. For example, an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a promoter sequence. A cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector. A cloning vector may be, e.g., a plasmid vector or a bacteriophage vector. A transfer vector may be a vector which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors. A vector in the context of the present invention may be, e.g., an RNA vector or a DNA vector. Preferably, a vector is a DNA molecule. For example, a vector in the sense of the present application comprises a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication. Preferably, a vector in the context of the present application is a plasmid vector.

Cells

Cells transformed with such vectors are also included within the scope of the invention. Examples of such cells include but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells or plant cells. In one embodiment the cells are mammalian, e.g., human, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells. Accordingly, the present invention also provides a cell expressing the antibody, or the antigen binding fragment thereof, according to the present invention; or comprising the vector according to the present invention.

In particular, the cell may be transfected with a vector according to the present invention, preferably with an expression vector. The term “transfection” refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, into cells, preferably into eukaryotic cells. In the context of the present invention, the term “transfection” encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, preferably into eukaryotic cells, such as into mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g. based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle based transfection, virus based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine etc. Preferably, the introduction is non-viral.

Polypeptides

The present invention also relates to a polypeptide, e.g. an isolated or purified immunogenic polypeptide, comprising at least two epitopes that specifically bind to the antibody, or the antigen binding fragment thereof, according to the present invention. For example, an (immunogenic) polypeptide according to the present invention may be used for a vaccine, in diagnosis of a disease as described herein and/or during production, purification and/or validation processes and/or during quality controls of an antibody according to the present invention. Preferably, the (immunogenic) polypeptide according to the present invention, which comprises at least two epitopes that specifically bind to the antibody, or the antigen binding fragment thereof, according to the present invention, is a recombinant polypeptide, i.e. a polypeptide which does not occur naturally.

Monoclonal and recombinant antibodies are particularly useful in identification and purification of the individual polypeptides or other antigens against which they are directed. The antibodies of the invention have thus additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). In these applications, the antibodies can be labeled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme. The antibodies may also be used for the molecular identification and characterization (epitope mapping) of antigens.

The polypeptides that bind to the antibodies of the present invention may thus have a number of uses. The polypeptides and polypeptide variants thereof in purified or synthetic form can be used to raise immune responses (i.e., as a vaccine, or for the production of antibodies for other uses) or for screening sera for antibodies that immunoreact with the epitope or mimotopes thereof. In one embodiment such polypeptides or polypeptide variants, or antigen comprising such polypeptides or polypeptide variants may be used as a vaccine for raising an immune response that comprises antibodies of the same quality as those described in the present invention.

The polypeptides that bind to the antibodies of the present invention may also be useful in screening for ligands that bind to said polypeptides. Such ligands, include but are not limited to antibodies; including those from camels, sharks and other species, fragments of antibodies, peptides, phage display technology products, aptamers, adnectins or fragments of other viral or cellular proteins, may block the epitope and so prevent infection. Such ligands are encompassed within the scope of the invention.

Optional Additional Features of the Antibodies

Antibodies of the invention may be coupled, for example, to a drug for delivery to a treatment site or coupled to a detectable label to facilitate imaging of a site comprising cells of interest. Methods for coupling antibodies to drugs and detectable labels are well known in the art, as are methods for imaging using detectable labels. Labeled antibodies may be employed in a wide variety of assays, employing a wide variety of labels. Detection of the formation of an antibody-antigen complex between an antibody of the invention and an epitope of interest on a cytokine, in particular GM-CSF, can be facilitated by attaching a detectable substance to the antibody. Suitable detection means include the use of labels such as radionuclides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substrates or co-factors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 1251, 1311, 35S, or 3H. Such labeled reagents may be used in a variety of well-known assays, such as radioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescent immunoassays, and the like. Labeled antibodies according to the present invention may be thus be used in such assays for example as described in U.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837; and 4,233,402.

An antibody according to the invention may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent, or a radioactive metal ion or radioisotope. Examples of radioisotopes include, but are not limited to, 1-131, 1-123, 1-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and the like. Such antibody conjugates can be used for modifying a given biological response; the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin.

Techniques for conjugating such therapeutic moiety to antibodies are well known. See, for example, Arnon et al. (1985) “Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy,” in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), pp. 243-256; ed. Hellstrom et al. (1987) “Antibodies for Drug Delivery,” in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker, Inc.), pp. 623-653; Thorpe (1985) “Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological and Clinical Applications, ed. Pinchera et al. pp. 475-506 (Editrice Kurtis, Milano, Italy, 1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” in Monoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin et al. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al. (1982) Immunol. Rev. 62:119-158.

Alternatively, an antibody, or antibody fragment thereof, can be conjugated to a second antibody, or antibody fragment thereof, to form an antibody heteroconjugate as described in U.S. Pat. No. 4,676,980. In addition, linkers may be used between the labels and the antibodies of the invention, e.g., as described in U.S. Pat. No. 4,831,175. Antibodies or, antigen-binding fragments thereof may be directly labeled with radioactive iodine, indium, yttrium, or other radioactive particle known in the art, e.g., as described in U.S. Pat. No. 5,595,721. Treatment may consist of a combination of treatment with conjugated and non-conjugated antibodies administered simultaneously or subsequently e.g., as described in WO00/52031; WO00/52473.

Antibodies of the invention may also be attached to a solid support. Additionally, antibodies of the invention, or functional antibody fragments thereof, can be chemically modified by covalent conjugation to a polymer to, for example, increase their circulating half-life. Examples of polymers, and methods to attach them to peptides, are shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546. In some embodiments the polymers may be selected from polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula: R(O—CH2-CH2)n O—R where R can be hydrogen, or a protective group such as an alkyl or alkanol group. Preferably, the protective group may have between 1 and 8 carbons. For example, the protective group is methyl. The symbol n is a positive integer. In one embodiment n is between 1 and 1,000. In another embodiment n is between 2 and 500. Preferably, the PEG has an average molecular weight between 1,000 and 40,000, more preferably the PEG has a molecular weight between 2,000 and 20,000, even more preferably the PEG has a molecular weight between 3,000 and 12,000. Furthermore, PEG may have at least one hydroxy group, for example the PEG may have a terminal hydroxy group. For example, it is the terminal hydroxy group which is activated to react with a free amino group on the inhibitor. However, it will be understood that the type and amount of the reactive groups may be varied to achieve a covalently conjugated PEG/antibody of the present invention.

Water-soluble polyoxyethylated polyols are also useful in the present invention. They include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), and the like. In one embodiment, POG is used. Without being bound by any theory, because the glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, triglycerides, this branching would not necessarily be seen as a foreign agent in the body. POG may have a molecular weight in the same range as PEG. Another drug delivery system that can be used for increasing circulatory half-life is the liposome. Methods of preparing liposome delivery systems are known to one of skill in the art. Other drug delivery systems are known in the art and are described in, for example, referenced in Poznansky et al. (1980) and Poznansky (1984).

Antibodies of the invention may be provided in purified form. Typically, the antibody will be present in a composition that is substantially free of other polypeptides e.g., where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.

Antibodies of the invention may be immunogenic in non-human (or heterologous) hosts e.g., in mice. In particular, the antibodies may have an idiotope that is immunogenic in non-human hosts, but not in a human host. Antibodies of the invention for human use include those that cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, etc. and cannot generally be obtained by humanization or from xeno-mice.

Production of Antibodies

Antibodies according to the invention can be made by any method known in the art. For example, the general methodology for making monoclonal antibodies using hybridoma technology is well known (Kohler, G. and Milstein, C. 1975; Kozbar et al. 1983). In one embodiment, the alternative EBV immortalization method described in WO2004/076677 is used.

Using the method described in WO 2004/076677, B cells producing the antibody of the invention can be transformed with EBV and a polyclonal B cell activator. Additional stimulants of cellular growth and differentiation may optionally be added during the transformation step to further enhance the efficiency. These stimulants may be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is added during the immortalization step to further improve the efficiency of immortalization, but its use is not essential. The immortalized B cells produced using these methods can then be cultured using methods known in the art and antibodies isolated therefrom.

Using the method described in WO 2010/046775, plasma cells can be cultured in limited numbers, or as single plasma cells in microwell culture plates. Antibodies can be isolated from the plasma cell cultures. Further, from the plasma cell cultures, RNA can be extracted and PCR can be performed using methods known in the art. The VH and VL regions of the antibodies can be amplified by RT-PCR (reverse transcriptase PCR), sequenced and cloned into an expression vector that is then transfected into HEK293T cells or other host cells. The cloning of nucleic acid in expression vectors, the transfection of host cells, the culture of the transfected host cells and the isolation of the produced antibody can be done using any methods known to one of skill in the art.

The antibodies may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Techniques for purification of antibodies, e.g., monoclonal antibodies, including techniques for producing pharmaceutical-grade antibodies, are well known in the art.

Fragments of the antibodies of the invention can be obtained from the antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction. Alternatively, fragments of the antibodies can be obtained by cloning and expression of part of the sequences of the heavy or light chains. Antibody “fragments” include Fab, Fab′, F(ab′)2 and Fv fragments. The invention also encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of an antibody of the invention. For example, the invention includes a scFv comprising the CDRs from an antibody of the invention. Also included are heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, as well as single chain antibodies, e.g., single chain Fv in which the heavy and light chain variable domains are joined by a peptide linker.

Antibody fragments of the invention may impart monovalent or multivalent interactions and be contained in a variety of structures as described above. For instance, scFv molecules may be synthesized to create a trivalent “triabody” or a tetravalent “tetrabody.” The scFv molecules may include a domain of the Fc region resulting in bivalent minibodies. In addition, the sequences of the invention may be a component of multispecific molecules in which the sequences of the invention target the epitopes of the invention and other regions of the molecule bind to other targets. Exemplary molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies (Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

Standard techniques of molecular biology may be used to prepare DNA sequences encoding the antibodies or antibody fragments of the present invention. Desired DNA sequences may be synthesized completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.

Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the antibody molecules of the present invention or fragments thereof. Bacterial, for example E. coli, and other microbial systems may be used, in part, for expression of antibody fragments such as Fab and F(ab′)2 fragments, and especially Fv fragments and single chain antibody fragments, for example, single chain Fvs. Eukaryotic, e.g., mammalian, host cell expression systems may be used for production of larger antibody molecules, including complete antibody molecules. Suitable mammalian host cells include, but are not limited to, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

The present invention also provides a process for the production of an antibody molecule according to the present invention comprising culturing a host cell comprising a vector encoding a nucleic acid of the present invention under conditions suitable for expression of protein from DNA encoding the antibody molecule of the present invention, and isolating the antibody molecule.

The antibody molecule may comprise only a heavy or light chain polypeptide, in which case only a heavy chain or light chain polypeptide coding sequence needs to be used to transfect the host cells. For production of products comprising both heavy and light chains, the cell line may be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector may be used, the vector including sequences encoding light chain and heavy chain polypeptides.

Alternatively, antibodies according to the invention may be produced by (i) expressing a nucleic acid sequence according to the invention in a host cell, and (ii) isolating the expressed antibody product. Additionally, the method may include (iii) purifying the isolated antibody. Transformed B cells and cultured plasma cells may be screened for those producing antibodies of the desired specificity or function.

The screening step may be carried out by any immunoassay, e.g., ELISA, by staining of tissues or cells (including transfected cells), by neutralization assay or by one of a number of other methods known in the art for identifying desired specificity or function. The assay may select on the basis of simple recognition of one or more antigens, or may select on the additional basis of a desired function e.g., to select neutralizing antibodies rather than just antigen-binding antibodies, to select antibodies that can change characteristics of targeted cells, such as their signaling cascades, their shape, their growth rate, their capability of influencing other cells, their response to the influence by other cells or by other reagents or by a change in conditions, their differentiation status, etc.

Individual transformed B cell clones may then be produced from the positive transformed B cell culture. The cloning step for separating individual clones from the mixture of positive cells may be carried out using limiting dilution, micromanipulation, single cell deposition by cell sorting or another method known in the art.

Nucleic acid from the cultured plasma cells can be isolated, cloned and expressed in HEK293T cells or other known host cells using methods known in the art.

The immortalized B cell clones or the transfected host-cells of the invention can be used in various ways e.g., as a source of monoclonal antibodies, as a source of nucleic acid (DNA or mRNA) encoding a monoclonal antibody of interest, for research, etc.

The invention also provides a composition comprising immortalized B memory cells or transfected host cells that produce antibodies according to the present invention.

The immortalized B cell clone or the cultured plasma cells of the invention may also be used as a source of nucleic acid for the cloning of antibody genes for subsequent recombinant expression. Expression from recombinant sources is more common for pharmaceutical purposes than expression from B cells or hybridomas e.g., for reasons of stability, reproducibility, culture ease, etc.

Thus the invention also provides a method for preparing a recombinant cell, comprising the steps of: (i) obtaining one or more nucleic acids (e.g., heavy and/or light chain mRNAs) from the B cell clone or the cultured plasma cells that encodes the antibody of interest; (ii) inserting the nucleic acid into an expression vector and (iii) transfecting the vector into a host cell in order to permit expression of the antibody of interest in that host cell.

Similarly, the invention provides a method for preparing a recombinant cell, comprising the steps of: (i) sequencing nucleic acid(s) from the B cell clone or the cultured plasma cells that encodes the antibody of interest; and (ii) using the sequence information from step (i) to prepare nucleic acid(s) for insertion into a host cell in order to permit expression of the antibody of interest in that host cell. The nucleic acid may, but need not, be manipulated between steps (i) and (ii) to introduce restriction sites, to change codon usage, and/or to optimize transcription and/or translation regulatory sequences.

Furthermore, the invention also provides a method of preparing a transfected host cell, comprising the step of transfecting a host cell with one or more nucleic acids that encode an antibody of interest, wherein the nucleic acids are nucleic acids that were derived from an immortalized B cell clone or a cultured plasma cell of the invention. Thus the procedures for first preparing the nucleic acid(s) and then using it to transfect a host cell can be performed at different times by different people in different places (e.g., indifferent countries).

These recombinant cells of the invention can then be used for expression and culture purposes. They are particularly useful for expression of antibodies for large-scale pharmaceutical production. They can also be used as the active ingredient of a pharmaceutical composition. Any suitable culture technique can be used, including but not limited to static culture, roller bottle culture, ascites fluid, hollow-fiber type bioreactor cartridge, modular minifermenter, stirred tank, microcarrier culture, ceramic core perfusion, etc.

Methods for obtaining and sequencing immunoglobulin genes from B cells or plasma cells are well known in the art (e.g., see Chapter 4 of Kuby Immunology, 4th edition, 2000).

The transfected host cell may be a eukaryotic cell, including yeast and animal cells, particularly mammalian cells (e.g., CHO cells, NS0 cells, human cells such as PER.C6 or HKB-11 cells, myeloma cells), as well as plant cells. Preferred expression hosts can glycosylate the antibody of the invention, particularly with carbohydrate structures that are not themselves immunogenic in humans. In one embodiment the transfected host cell may be able to grow in serum-free media. In a further embodiment the transfected host cell may be able to grow in culture without the presence of animal-derived products. The transfected host cell may also be cultured to give a cell line.

The invention also provides a method for preparing one or more nucleic acid molecules (e.g., heavy and light chain genes) that encode an antibody of interest, comprising the steps of: (i) preparing an immortalized B cell clone or culturing plasma cells according to the invention; (ii) obtaining from the B cell clone or the cultured plasma cells nucleic acid that encodes the antibody of interest. Further, the invention provides a method for obtaining a nucleic acid sequence that encodes an antibody of interest, comprising the steps of: (i) preparing an immortalized B cell clone or culturing plasma cells according to the invention; (ii) sequencing nucleic acid from the B cell clone or the cultured plasma cells that encodes the antibody of interest.

The invention further provides a method of preparing nucleic acid molecule(s) that encode an antibody of interest, comprising the step of obtaining the nucleic acid that was obtained from a transformed B cell clone or cultured plasma cells of the invention. Thus the procedures for first obtaining the B cell clone or the cultured plasma cell, and then obtaining nucleic acid(s) from the B cell clone or the cultured plasma cells can be performed at different times by different people in different places (e.g., in different countries).

The invention also comprises a method for preparing an antibody (e.g., for pharmaceutical use) according to the present invention, comprising the steps of: (i) obtaining and/or sequencing one or more nucleic acids (e.g., heavy and light chain genes) from the selected B cell clone or the cultured plasma cells expressing the antibody of interest; (ii) inserting the nucleic acid(s) into or using the nucleic acid(s) sequence(s) to prepare an expression vector; (iii) transfecting a host cell that can express the antibody of interest; (iv) culturing or sub-culturing the transfected host cells under conditions where the antibody of interest is expressed; and, optionally, (v) purifying the antibody of interest.

The invention also provides a method of preparing an antibody comprising the steps of culturing or sub-culturing a transfected host cell population under conditions where the antibody of interest is expressed and, optionally, purifying the antibody of interest, wherein said transfected host cell population has been prepared by (i) providing nucleic acid(s) encoding a selected antibody of interest that is produced by a B cell clone or cultured plasma cells prepared as described above, (ii) inserting the nucleic acid(s) into an expression vector, (iii) transfecting the vector in a host cell that can express the antibody of interest, and (iv) culturing or sub-culturing the transfected host cell comprising the inserted nucleic acids to produce the antibody of interest. Thus the procedures for first preparing the recombinant host cell and then culturing it to express antibody can be performed at very different times by different people in different places (e.g., in different countries).

Pharmaceutical Compositions

The present invention also provides a pharmaceutical composition comprising one or more of:

-   (i) the antibody, or antibody fragments according to the present     invention; -   (ii) the nucleic acid encoding the antibody, or antibody fragments     according to the present invention; -   (iii) the vector encoding the nucleic acid according to the present     invention; -   (iv) the cell expressing the antibody according to the present     invention or comprising the vector according to the present     invention; or -   (v) the immunogenic polypeptide recognized by the antibodies or     antigen binding fragment thereof according to the present invention.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier, diluent and/or excipient. Preferably, the pharmaceutical composition according to the present invention comprises one or more of:

-   (i) the antibody, or antibody fragments according to the present     invention; -   (ii) the nucleic acid encoding the antibody, or antibody fragments     according to the present invention; -   (iii) the vector encoding the nucleic acid according to the present     invention; -   (iv) the cell expressing the antibody according to the present     invention or comprising the vector according to the present     invention; or -   (v) the immunogenic polypeptide recognized by the antibodies or     antigen binding fragment thereof according to the present invention;     and     a pharmaceutically acceptable excipient, diluent and/or carrier.

Although the carrier or excipient may facilitate administration, it should not itself induce the production of antibodies harmful to the individual receiving the composition. Nor should it be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.

Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.

Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the subject.

Compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., a lyophilized composition, like Synagis™ and Herceptin™, for reconstitution with sterile water containing a preservative). The composition may be prepared for topical administration e.g., as an ointment, cream or powder. The composition may be prepared for oral administration e.g., as a tablet or capsule, as a spray, or as a syrup (optionally flavored). The composition may be prepared for pulmonary administration e.g., as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g., as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a subject. For example, a lyophilized antibody can be provided in kit form with sterile water or a sterile buffer.

It is preferred that the active ingredient in the composition is an antibody molecule, an antibody fragment or variants and derivatives thereof, in particular the active ingredient in the composition is an antibody, an antibody fragment or variants and derivatives thereof, according to the present invention. As such, it may be susceptible to degradation in the gastrointestinal tract. Thus, if the composition is to be administered by a route using the gastrointestinal tract, the composition may contain agents which protect the antibody from degradation but which release the antibody once it has been absorbed from the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

Pharmaceutical compositions of the invention generally have a pH between 5.5 and 8.5, in some embodiments this may be between 6 and 8, and in other embodiments about 7. The pH may be maintained by the use of a buffer. The composition may be sterile and/or pyrogen free. The composition may be isotonic with respect to humans. In one embodiment pharmaceutical compositions of the invention are supplied in hermetically-sealed containers.

Within the scope of the invention are compositions present in several forms of administration; the forms include, but are not limited to, those forms suitable for parenteral administration, e.g., by injection or infusion, for example by bolus injection or continuous infusion. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilizing and/or dispersing agents. Alternatively, the antibody molecule may be in dry form, for reconstitution before use with an appropriate sterile liquid. A vehicle is typically understood to be a material that is suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound, in particular the antibodies according to the present invention. For example, the vehicle may be a physiologically acceptable liquid, which is suitable for storing, transporting, and/or administering a pharmaceutically active compound, in particular the antibodies according to the present invention. Once formulated, the compositions of the invention can be administered directly to the subject. In one embodiment the compositions are adapted for administration to mammalian, e.g., human subjects.

The pharmaceutical compositions of this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal or rectal routes. Hyposprays may also be used to administer the pharmaceutical compositions of the invention. Preferably, the pharmaceutical composition may be prepared for oral administration, e.g. as tablets, capsules and the like, for topical administration, or as injectable, e.g. as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.

For injection, e.g. intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will preferably be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required. Whether it is a polypeptide, peptide, or nucleic acid molecule, other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. For injection, the pharmaceutical composition according to the present invention may be provided for example in a pre-filled syringe.

The inventive pharmaceutical composition as defined above may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added.

For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient, i.e. the inventive transporter cargo conjugate molecule as defined above, is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

The inventive pharmaceutical composition may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g. including diseases of the skin or of any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the inventive pharmaceutical composition may be formulated in a suitable ointment, containing the inventive pharmaceutical composition, particularly its components as defined above, suspended or dissolved in one or more carriers. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the inventive pharmaceutical composition can be formulated in a suitable lotion or cream. In the context of the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Dosage treatment may be a single dose schedule or a multiple dose schedule, whereby in the context of the present invention a multiple dose schedule is preferred. Known antibody-based pharmaceuticals, in particular anti-cytokine, e.g. anti-GM-CSF, based pharmaceuticals provide guidance relating to frequency of administration e.g., whether a pharmaceutical should be delivered daily, weekly, monthly, etc. Frequency and dosage may also depend on the severity of symptoms.

For example, the pharmaceutical composition according to the present invention may be administered daily, e.g. once or several times per day, e.g. once, twice, three times or four times per day, preferably once or twice per day, more preferable once per day, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 or more days, e.g. daily for 1, 2, 3, 4, 5, 6 months. Preferably, the pharmaceutical composition according to the present invention may be administered weekly, e.g. once or twice, preferably once per week, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 or more weeks, e.g. weekly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or weekly for 2, 3, 4, or 5 years.

In particular, it is preferred that for a single dose, e.g. a daily, weekly or monthly dose, preferably for a weekly dose, the amount of the antibody, or the antigen binding fragment thereof, in the pharmaceutical composition according to the present invention, does not exceed 150 mg, preferably does not exceed 100 mg, more preferably does not exceed 50 mg, even more preferably does not exceed 20 mg, and particularly preferably does not exceed 10 mg. This amount of antibody preferably refers to a single dose as described above, which is for example administered daily, weekly etc. as described above. Such a low amount of the antibody according to the present invention could be produced and formulated in a stable form (e.g., in a lyophilized formulation, where for instance previous studies have shown that monoclonal antibodies preserved by lyophilization are stable for 33 months at 40° C. and 5 months at 50° C.) and at an affordable cost.

Pharmaceutical compositions typically include an effective amount of one or more antibodies of the invention and/or a polypeptide comprising an epitope that binds an antibody of the invention i.e., an amount that is sufficient to treat, ameliorate, attenuate or prevent a desired disease or condition, or to exhibit a detectable therapeutic effect. Therapeutic effects also include reduction or attenuation in pathogenic potency or physical symptoms. The precise effective amount for any particular subject will depend upon their size, weight, and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. The effective amount for a given situation is determined by routine experimentation and is within the judgment of a clinician. For purposes of the present invention, an effective dose will generally be from about 0.005 to about 100 mg/kg, preferably from about 0.0075 to about 50 mg/kg, more preferably from about 0.01 to about 10 mg/kg, even more preferably from about 0.02 to about 5 mg/kg, and particularly preferably from about 0.03 to about 1 mg/kg of the antibody of the present invention (e.g. amount of the antibody in the pharmaceutical composition) in relation to the bodyweight (e.g., in kg) of the individual to which it is administered.

Moreover, the pharmaceutical composition according to the present invention may also comprises at least two antibodies or antigen binding fragments thereof, according to the present invention, wherein the two antibodies, or the antigen binding fragments thereof, specifically bind to a different set of non-overlapping epitopes on the cytokine, in particular on GM-CSF. For example, the pharmaceutical composition according to the present invention comprises a first antibody or an antigen binding fragment thereof, according to the present invention, and a second antibody, or an antigen binding fragment thereof, according to the present invention, wherein the first antibody, or the antigen binding fragment thereof, specifically binds to a first set of non-overlapping epitopes on the cytokine, in particular on GM-CSF, which is different from the set of non-overlapping epitopes on the cytokine, in particular on GM-CSF to which the second antibody or the second antigen binding fragment thereof binds. “Different sets of non-overlapping epitopes on the cytokine, in particular on GM-CSF” means herein that for example a first set includes an epitope I and an epitope II on a cytokine and a second (different) set includes an epitope I and an epitope III on a cytokine. Thus, the different sets may be overlapping, i.e. comprise the same epitopes, however they usually differ in at least one epitope. Preferably, however, the different sets of epitopes are non-overlapping, i.e. the first antibody binds to at least two different sites on a cytokine, e.g. GM-CSF, wherein each of the at least two different sites is different from the at least two different sites on a cytokine, e.g. GM-CSF, to which the second antibody binds. For example, if the pharmaceutical composition according to the present invention comprises two antibodies or antigen binding fragments thereof, according to the present invention, and the first antibody, or the antigen binding fragment thereof, specifically binds to non-overlapping sites I and II on the cytokine, in particular on GM-CSF, the second antibody, or the antigen binding fragment thereof, may specifically bind to non-overlapping sites III and IV on the cytokine, in particular GM-CSF. In such a way all (non-overlapping) sites on a cytokine, e.g. GM-CSF, may be covered by antibodies according to the present invention.

Moreover, the pharmaceutical composition may also contain more than two, e.g. 3, 4, 5, 6, etc., antibodies according to the present invention, whereby at least two, preferably more than two, more preferably all antibodies contained, bind to different sets of epitopes on the cytokine, e.g. GM-CSF.

Preferably, the two antibodies according to the present invention are present in the pharmaceutical composition at equimolar amounts, preferably as an equimolar mixture.

Preferably, compositions can include two or more (e.g., 2, 3, 4, 5 etc.) antibodies of the invention to provide an additive or synergistic therapeutic effect. The term “synergy” is used to describe a combined effect of two or more active agents that is greater than the sum of the individual effects of each respective active agent. Thus, where the combined effect of two or more agents results in “synergistic inhibition” of an activity or process, it is intended that the inhibition of the activity or process is greater than the sum of the inhibitory effects of each respective active agent. The term “synergistic therapeutic effect” refers to a therapeutic effect observed with a combination of two or more therapies wherein the therapeutic effect (as measured by any of a number of parameters) is greater than the sum of the individual therapeutic effects observed with the respective individual therapies.

In another embodiment, the composition may comprise one or more (e.g., 2, 3, etc.) antibodies according the invention and one or more (e.g., 2, 3, etc.) additional antibodies against the cytokine, in particular GM-CSF. Further, the administration of antibodies of the invention together with antibodies specific to other cytokines or, more generally, to other antigens, are within the scope of the invention. The antibodies of the invention can be administered either combined/simultaneously or at separate times from antibodies specific to other cytokines or, more generally, to other antigens.

Examples of antibodies of the invention against a cytokine, in particular against GM-CSF, include, but are not limited to, Ts1GC1, Ts1GC2a, Ts2GC2b, Ts2GC2c, Ts3GC2d, Ts3GC2e, Bs3GC1a, Bs3GC1b, Bs2GC1c, Bs2GC1d, Bs1GC2a, Bs3GC2b, Bs1GC3a, Bs3GC3b, Bs3GC4, and Bs3GC5.

Moreover, a pharmaceutical composition comprising the antibody according to gTs1GC1 or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gTs1GC2a or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gTs2GC2b or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gTs2GC2c or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gTs3GC2d or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gTs3GC2e or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gBs3GC1a or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gBs3GC1b or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gBs2GC1c or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gBs2GC1d or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gBs1GC2a or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gBs3GC2b or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gBs1GC3a or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gBs3GC3b or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gBs3GC4 or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody according to gBs3GC5 or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred.

In addition, a pharmaceutical composition comprising the antibody Ts1GC1 or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Ts1GC2a or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Ts2GC2b or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Ts2GC2c or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Ts3GC2d or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the Ts3GC2e or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Bs3GC1a or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Bs3GC1b or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Bs2GC1c or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Bs2GC1d or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Bs1GC2a or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Bs3GC2b or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Bs1GC3a or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Bs3GC3b or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Bs3GC4 or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred. A pharmaceutical composition comprising the antibody Bs3GC5 or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier is also preferred.

In one embodiment, a composition of the invention may include antibodies of the invention, wherein the antibodies may make up at least 50% by weight (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) of the total protein in the composition. In such a composition, the antibodies are preferably in purified form.

The present invention also provides a method of preparing a pharmaceutical composition comprising the steps of (i) preparing an antibody of the invention; and (ii) admixing the purified antibody with one or more pharmaceutically-acceptable carriers.

In another embodiment, a method of preparing a pharmaceutical composition comprises the step of: admixing an antibody with one or more pharmaceutically-acceptable carriers, wherein the antibody is a monoclonal antibody that was obtained from a transformed B cell or a cultured plasma cell of the invention. Thus the procedures for first obtaining the monoclonal antibody and then preparing the pharmaceutical can be performed at very different times by different people in different places (e.g., in different countries).

As an alternative to delivering antibodies or B cells for therapeutic purposes, it is possible to deliver nucleic acid (typically DNA) that encodes the monoclonal antibody (or active fragment thereof) of interest derived from the B cell or the cultured plasma cells to a subject, such that the nucleic acid can be expressed in the subject in situ to provide a desired therapeutic effect. Suitable gene therapy and nucleic acid delivery vectors are known in the art.

Compositions may include an antimicrobial, particularly if packaged in a multiple dose format. They may comprise detergent e.g., a Tween (polysorbate), such as Tween 80. Detergents are generally present at low levels e.g., less than 0.01%. Compositions may also include sodium salts (e.g., sodium chloride) to give tonicity. For example, a concentration of 10±2 mg/ml NaCl is typical.

Further, compositions may comprise a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose) e.g., at around 15-30 mg/ml (e.g., 25 mg/ml), particularly if they are to be lyophilized or if they include material which has been reconstituted from lyophilized material. The pH of a composition for lyophilisation may be adjusted to between 5 and 8, or between 5.5 and 7, or around 6.1 prior to lyophilisation.

The compositions of the invention may also comprise one or more immunoregulatory agents. In one embodiment, one or more of the immunoregulatory agents include(s) an adjuvant.

Medical Treatments and Uses

In a further aspect, the present invention provides the use of an antibody, or an antigen binding fragment thereof, according to the present invention, the nucleic acid according to the present invention, the vector according to the present invention, the cell according to the present invention, the immunogenic polypeptide according to the present invention, or the pharmaceutical composition according to the present invention in (i) prophylaxis, treatment or attenuation of inflammatory and/or autoimmune diseases; or in (ii) diagnosis of inflammatory and/or autoimmune diseases.

Inflammatory diseases may be due to a variety of causes. In the context of the present invention preferably such inflammatory diseases may be treated, attenuated and/or prevented, which are due to physical causes, e.g. burns, frostbite, physical injury, blunt or penetrating, foreign bodies including splinters, dirt and debris, trauma and ionizing radiation; biological causes, e.g. infection by pathogens, immune reactions due to hypersensitivity, and stress; and chemical causes, e.g. chemical irritants, toxins, and alcohol.

Inflammatory diseases (also referred to as inflammatory disorders) include the following diseases and, thus, in the context of the present invention an inflammatory disease may be preferably selected from the group consisting of appendicitis, bursitis, colitis, cystitis, dermatitis, phlebitis, RSD/CRPS, rhinitis, tendonitis, tonsillitis, vasculitis, Alzheimer's disease, ankylosing spondylitis, arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis, asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematous (SLE), nephritis, Parkinson's disease, ulcerative colitis, Acne vulgaris, autoinflammatory diseases, celiac disease, prostatitis, pulmonary alveolar proteinosis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, vasculitis, interstitial cystitis, inflammatory myopathies, encephalomyelitis, in particular acute disseminated encephalomyelitis, spondylitis, in particular ankylosing spondylitis, antisynthetase syndrome, dermatitis, in particular atopic dermatitis or contact dermatitis, hepatitis, in particular autoimmune hepatitis, autoimmune peripheral neuropathy, pancreatitis, in particular autoimmune pancreatitis, Behçet's disease, Bickerstaffs, encephalitis, Blau syndrome, Coeliac disease, Chagas disease, polyneuropathy, in particular chronic inflammatory demyelinating polyneuropathy, osteomyelitis, in particular chronic recurrent multifocal osteomyelitis, Churg-Strauss syndrome, Cogan syndrome, giant-cell arteritis, CREST syndrome, vasculitis, in particular cutaneous small-vessel vasculitis and urticarial vasculitis, dermatitis herpetiformis, dermatomyositis, systemic scleroderma, Dressler's syndrome, drug-induced lupus erythematosus, discoid lupus erythematosus, enthesitis, eosinophilic fasciitis, eosinophilic gastroenteritis, erythema nodosum, Idiopathic pulmonary fibrosis, gastritis, Grave's disease, Guillain-barre syndrome, Hashimoto's thyroiditis, Henoch-Schonlein purpura, Hidradenitis suppurativa, Idiopathic inflammatory demyelinating diseases, myositis, in particular inclusion body myositis, cystitis, in particular interstitial cystitis, Kawasaki disease, Lichen planus, lupoid hepatitis, Majeed syndrome, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, myelitis, in particular neuromyelitis optica, thyroiditis, in particular Ord's thyroiditis, rheumatism, in particular palindromic rheumatism, Parsonage-Turner syndrome, pemphigus vulgaris, perivenous encephalomyelitis, polyarteritis nodosa, polymyalgia, in particular polymyalgia rheumatica, polymyositis, cirrhosis, in particular primary biliary cirrhosis, cholangitis, in particular primary sclerosing cholangitis, progressive inflammatory neuropathy, Rasmussen's encephalitis, relapsing polychondritis, arthritis, in particular reactive arthritis (Reiter disease) and rheumatoid arthritis, rheumatic fever, sarcoidosis, Schnitzler syndrome, serum sickness, spondyloarthropathy, Takayasu's arteritis, Tolosa-Hunt syndrome, transverse myelitis, and Wegener's granulomatosis.

Autoimmune disorders (also referred to as autoimmune diseases) include the following diseases and, thus, in the context of the present invention an autoimmune disease may be preferably selected from the group consisting of autoimmune diseases of the CNS, auto-inflammatory diseases, Celiac disease; Sjogren's syndrome, systemic lupus erythematosus, Blau syndrome, Bullous pemphigoid, Cancer, Castleman's disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy, Chronic recurrent multifocal osteomyelitis, chronic obstructive pulmonary disease, Churg-Strauss syndrome, Cicatricial pemphigoid, Cogan syndrome, Cold agglutinin disease, Complement component 2 deficiency, Contact dermatitis, Cranial arteritis, CREST syndrome, Crohn's disease, Cushing's Syndrome, Dercum's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type 1, Diffuse cutaneous systemic sclerosis, Dressler's syndrome, lupus, Discoid lupus erythematosus, Eczema, Acute disseminated encephalomyelitis (ADEM), Addison's disease, Agammaglobulinemia, Amyotrophic lateral sclerosis (Also Lou Gehrig's disease; Motor Neuron Disease), Ankylosing Spondylitis Antiphospholipid syndrome, Antisynthetase syndrome, Atopic dermatitis, Autoimmune aplastic anemia, Autoimmune cardiomyopathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome, Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome, Autoimmune progesterone dermatitis, Autoimmune thrombocytopenic purpura, Autoimmune urticarial, Autoimmune uveitis, Balo disease/Balo concentric sclerosis, Behcet's disease, Berger's disease, Bickerstaffs encephalitis, Endometriosis, Enthesitis-related arthritis, Eosinophilic gastroenteritis, Epidermolysis bullosa acquisita, Erythroblastosis fetalis, Evan's syndrome, Fibrodysplasia ossificans, Fibrosing alveolitis (or Idiopathic pulmonary fibrosis), Gastritis, Glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's encephelopathy, Hashimoto's thyroiditis, Gestational Pemphigoid, Hidradenitis suppurativa, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (Autoimmune thrombocytopenic purpura), lgA nephropathy, Occular cicatricial pemphigoid, Inclusion body myositis, Rheumatoid arthritis, Chronic inflammatory Rheumatic fever, demyelinating polyneuropathy, Sarcoidosis, Palindromic rheumatism, Interstitial cystitis, Juvenile idiopathic Schizophrenia, PANDAS (pediatric arthritis aka Juvenile autoimmune rheumatoid arthritis), Schmidt syndrome, neuropsychiatric Kawasaki's disease another form of APS, Schnitzler syndrome, Paraneoplastic cerebellar myasthenic syndrome, Leukocytoclastic Serum Sickness, Lichen planus, Sjogren's syndrome, Lichen sclerosus, Parsonage-Tumer, Linear IgA disease, Still's disease, Pemphigus vulgaris, Lupoid hepatitis, Autoimmune hepatitis, Stiff person syndrome, Pemicious anaemia, Subacute bacterial endocarditis (SBE), POEMS syndrome, Lupus erythematosus, Sweet's syndrome, Sympathetic ophthalmia, Meniere's disease, Systemic lupus, Primary biliary cirrhosis, Miller-Fisher syndrome, Takayasu's arteritis, cholangitis, Progressive inflammatory neuropathy, Mucha-Habermann disease, Psoriasis, Psoriatic arthritis, Pyoderma gangrenosum, Multiple sclerosis, Pure red cell aplasia, Rasmussen's encephalitis, Myasthenia gravis, Transverse myelitis, Raynaud phenomenon, Microscopic colitis, Ulcerative colitis, Myositis, idiopathic inflammatory bowel disease (IBD), Neuromyelitis optica, Devic's disease, and Neuromyotonia.

Typically, autoimmune diseases arise from an abnormal immune response of the body against substances and tissues normally present in the body (autoimmunity). This may be restricted to certain organs (e.g. in autoimmune thyroiditis) or may involve a particular tissue in different places (e.g. Goodpasture's disease which may affect the basement membrane in both the lung and the kidney). Autoimmune diseases may be classified by corresponding type of hypersensitivity: type I (i.e. urticaria induced by autologous serum), type II, type III, or type IV.

In particular, at least some autoimmune disorders may also be inflammatory diseases and vice versa.

Autoimmune diseases and/or inflammatory diseases, which are preferably treated, prevented and/or attenuated in the context of the present invention, include multiple sclerosis, pulmonary alveolar proteinosis, arthritis, in particular rheumatoid arthritis, and asthma.

Within the scope of the invention are several forms and routes of administration of the antibody, or the antigen binding fragment thereof, the nucleic acid, the vector, the cell, the immunogenic polypeptide, or the pharmaceutical composition, as described above, in respect to the pharmaceutical composition. This applies also in the context of the use of the antibody, or the antigen binding fragment thereof, the nucleic acid, the vector, the cell, the immunogenic polypeptide as described herein, in particular regarding preferred forms and routes of administration.

Methods of diagnosis may include contacting an antibody or an antibody fragment with a sample. Such samples may be isolated from a subject, for example an isolated tissue sample taken from, for example, nasal passages, sinus cavities, salivary glands, lung, liver, pancreas, kidney, ear, eye, placenta, alimentary tract, heart, ovaries, pituitary, adrenals, thyroid, brain, skin or blood, preferably serum. The methods of diagnosis may also include the detection of an antigen/antibody complex, in particular following the contacting of an antibody or an antibody fragment with a sample. Such a detection step is typically performed at the bench, i.e. without any contact to the human or animal body. Examples of detection methods include e.g. ELISA (enzyme-linked immunosorbent assay).

The invention also provides the use of (i) an antibody, an antibody fragment, or variants and derivatives thereof according to the invention, (ii) an immortalized B cell clone according to the invention, (iii) a nucleic acid or a vector according to the present invention or (iv) a pharmaceutical composition of the invention in (a) the manufacture of a medicament for the treatment or attenuation of inflammatory and/or autoimmune diseases or (b) diagnosis of inflammatory and/or autoimmune diseases.

The invention also provides a composition of the invention for use as a medicament for the prevention or treatment of inflammatory and/or autoimmune diseases. It also provides the use of an antibody of the invention and/or a protein comprising an epitope to which such an antibody binds in the manufacture of a medicament for treatment of a subject and/or diagnosis in a subject. It also provides a method for treating a subject, comprising the step of administering to the subject a composition of the invention. In some embodiments the subject may be a human. One way of checking efficacy of therapeutic treatment involves monitoring disease symptoms after administration of the composition of the invention. Treatment can be a single dose schedule or a multiple dose schedule.

In one embodiment, an antibody, antibody fragment, immortalized B cell clone, or pharmaceutical composition according to the invention is administered to a subject in need of such treatment. Such a subject includes, but is not limited to, one who is particularly at risk of or susceptible to inflammatory and/or autoimmune diseases.

Antibodies and fragments thereof as described in the present invention may also be used in a kit for the diagnosis of inflammatory and/or autoimmune diseases. Further, at least two epitopes, in particular of a cytokine, e.g. GM-CSF, capable of binding an antibody of the invention may be used in a kit for monitoring the efficacy of application procedures by detecting the presence or determining the titer of the protective anti-cytokine, in particular anti-GM-CSF, antibodies.

The invention also provides a method of preparing a pharmaceutical, comprising the step of admixing a monoclonal antibody with one or more pharmaceutically-acceptable carriers, wherein the monoclonal antibody is a monoclonal antibody that was obtained from a transfected host cell of the invention. Thus the procedures for first obtaining the monoclonal antibody (e.g., expressing it and/or purifying it) and then admixing it with the pharmaceutical carrier(s) can be performed at very different times by different people in different places (e.g., in different countries).

Starting with a transformed B cell or a cultured plasma cell of the invention, various steps of culturing, sub-culturing, cloning, sub-cloning, sequencing, nucleic acid preparation etc. can be performed in order to perpetuate the antibody expressed by the transformed B cell or the cultured plasma cell, with optional optimization at each step. In one embodiment, the above methods further comprise techniques of optimization (e.g., affinity maturation or optimization) applied to the nucleic acids encoding the antibody. The invention encompasses all cells, nucleic acids, vectors, sequences, antibodies etc. used and prepared during such steps.

In all these methods, the nucleic acid used in the expression host may be manipulated to insert, delete or alter certain nucleic acid sequences. Changes from such manipulation include, but are not limited to, changes to introduce restriction sites, to amend codon usage, to add or optimize transcription and/or translation regulatory sequences, etc. It is also possible to change the nucleic acid to alter the encoded amino acids. For example, it may be useful to introduce one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions and/or insertions into the antibody's amino acid sequence. Such point mutations can modify effector functions, antigen-binding affinity, post-translational modifications, immunogenicity, etc., can introduce amino acids for the attachment of covalent groups (e.g., labels) or can introduce tags (e.g., for purification purposes). Mutations can be introduced in specific sites or can be introduced at random, followed by selection (e.g., molecular evolution). For instance, one or more nucleic acids encoding any of the CDR regions, heavy chain variable regions or light chain variable regions of antibodies of the invention can be randomly or directionally mutated to introduce different properties in the encoded amino acids. Such changes can be the result of an iterative process where initial changes are retained and new changes at other nucleotide positions are introduced. Further, changes achieved in independent steps may be combined. Different properties introduced into the encoded amino acids may include, but are not limited to, enhanced affinity.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the description and accompanying figures. Such modifications fall within the scope of the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control.

The following Figures, Sequences and Examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

DESCRIPTION OF FIGURES

FIG. 1 shows (A) SPR cross-competition between GCA21, GCA7 and GCB59, (B) A multichannel chip coated with GCA21, GCA7 or site IV GCB59 was saturated with GM-CSF and serially exposed to an excess of the same antibodies. (C) SEC-HPLC profile of samples containing the three non-cross-competing antibodies, alone or with GM-CSF added in equimolar concentrations (1:1) or in 10-fold antibody excess (10:1).

FIG. 2 shows the potent in vitro neutralization of GM-CSF by a combination of three antibodies. A fixed amount of GM-CSF (final concentration 50 pg ml-1) was incubated with serial dilutions of one or more antibodies, added to TF1 cells (10,000 per well) and cell proliferation was measured on day 3 by thymidine incorporation. (A) Scheme of the TF-1 bioassay. (B) Serial dilutions of single monoclonal antibodies or mixtures of two and three non-cross-competing antibodies were tested for their capacity to neutralize GM-CSF. (C) The sensitivity of the test was changed by varying the number of cells and the concentration of GM-CSF as indicated. Shown is for each experimental condition the inhibition obtained using single antibodies or a combination of three non-cross-competing antibodies.

FIG. 3 shows the Fc-dependent clearance of GM-CSF immune complexes in vivo. (A) A sandwich ELISA to detect GM-CSF in the presence of specific antibodies. A fixed amount of GM-CSF was added to mouse serum together with three monoclonal antibodies (GCA21, GCA7, GCB59) added separately or in combination. The quantification of GM-CSF was performed by a sandwich ELISA using an antibody specific for site II for capture and site I for detection. Serial dilutions of serum in neutral (left) or alkaline buffer (right) were added and GM-CSF concentration was determined with reference to a GM-CSF standard. The dotted line represents the concentration of GM-CSF measured in the absence of antibodies. (B) Female Balb/c mice (5 per group) were injected with 100 μg of monoclonal antibody, either GCA21 (1 mAb) or GCA21+GCA7+GCB59 (3 mAbs) in the IgG or IgG-LALA format, or with 2 mg total IgG from a PAP patient, followed by 2 μg GM-CSF after 16 hours. Sera were collected after 1 or 5 days and GM-CSF concentrations were measured by ELISA in untreated serum and in serum treated at pH 11.6 to dissociate immune complexes. Shown is the GM-CSF concentration on day 1 and on day 5 in untreated serum (left) or alkaline-treated serum (right). (C) Proliferation of TF-1 cells in response to different dilutions of serum of mice injected 24 hours before with GM-CSF and the indicated antibodies. (D) Binding of GM-CSF immune complexes formed by one or three antibodies (in the IgG1 or IgG1-LALA format) to TZM-b1 cells expressing Fc□RIIa or Fc□□RIIb, as measured by flow cytometry using an anti-IgG Fc specific antibody.

FIG. 4 shows the scheme of three bispecific construct types Bs1, Bs2 and Bs3 and of the three trispecific construct types Ts1, Ts2 and Ts3. Positions A, B, and/or C (if applicable) are shown for each of the construct types. Single chain variable domains (ScVd) of both heavy chain and light chain of different GM-CSF antibodies are added at the N-terminus and/or at the C-terminus of the heavy chain or the light chain of one GM-CSF antibody used as scaffold. Black ovals represent VH domains while dark grey ovals represent VL domains. The VH and VL of the different ScVds are joined together through specific linkers. Other linkers are used to join the ScVds between one another and to the full antibody used as scaffold. Light gray ovals represent IgG1 CH and CL domains.

FIG. 5 shows that TsGC1 binds to GM-CSF with very high affinity with a very slow off-rate (A). (B-C) TsGC1 can use all the 3 different specificities for binding to GM-CSF. GCA21, GCA7 and GCB59 were immobilized on a SPR chip and were serially exposed to GM-CSF followed by GCA7, GCB59 and GCA21, respectively, and finally by TsGC1. (E) TsGC1 can form high molecular weight complexes with GM-CSF. TsGC1 was immobilized on an SPR chip and serially exposed for 3 rounds to GM-CSF followed by soluble TsGC1. Shown is the same experiment performed with GCA7 as control.

FIG. 6 shows the extremely potent neutralization of GM-CSF by TsgC2d and BsGC3a as compared to single antibodies or combinations of antibodies forming TsgC2d and BsGC3a.

FIG. 7 shows the binding of immune complexes formed by GM-CSF and TsGC2d or BsGC3a to TZM-b1 cells expressing FcγRIIa or FcγRIIb, as measured by flow cytometry using an anti-IgG Fc specific antibody. The binding is compared to that of single antibodies and combinations of the 2 or 3 antibodies forming BsGC3a and TsGC2d, respectively.

EXAMPLES

Exemplary embodiments of the present invention are provided in the following examples. The following examples are presented only by way of illustration and to assist one of ordinary skill in using the invention. The examples are not intended in any way to otherwise limit the scope of the invention.

Example 1: Isolation and Characterization GM-CSF-Specific Antibodies

Peripheral blood samples were collected from five pulmonary alveolar proteinosis (PAP) patients. IgG memory B cells were isolated from cryopreserved or fresh PBMCs by a combination of magnetic and fluorescence-activated cell sorting, in particular using anti-FITC microbeads (Miltenyi Biotec) following staining of PBMCs with CD22-FITC (BD Phamingen). IgG memory B cells were then immortalized in clonal conditions with EBV (Epstein-Barr virus) and CpG in 384 well micro-plates in the presence of feeder cells as described by Traggiai E. et al. (2004) Nat Med. 10(8):871-5 and WO 2004/076677 A2. The culture supernatants were screened for the presence of GM-CSF-specific IgG antibodies by ELISA. Four immortalized B cell clones that produced GM-CSF monoclonal antibodies were identified. cDNA was synthesized from positive cultures and the antibody V genes (heavy chain and light chain variable regions) were sequenced and analyzed using the IMGT database (http://www.imgt.org/). The V(D)J gene usage of the four PAP autoantibodies is shown in Table 5.

TABLE 5 V(D)J gene usage of the 4 PAP autoantibodies. The antibodies use different V, D and J genes and are somatically mutated. The load of somatic mutations was comparable to that characteristic of T-cell-dependent responses against non-self-antigens, ranging from 8.8% to 16.7% in the VH gene segment and from 0% to 8.6% in the VL gene segment. mAb Heavy chain VDJ genes (% identity to GL) Light chain VJ genes (% identity to GL) GCA7 VH3-66 (91.2) D3-10 JH4 (93.8) VK4-1 (96)   JK3 (100)   GCA21 FH3-30-3 (83.3) D2-15 JH2 (84.9) VK1-5 (92.8) JK4 (97.2) GCB59 FH3-21 (86.8) D2-15 JH6 (77.4) VL3-21 (92.1) JL2 (91.9) GCE536 FH1-46 (87.9) D2-2 JH6 (85.5) FK3-20 (91.5) JK2 (92.1)

All antibodies were recombinantly produced as IgG1 by transient transfection of HEK 293 Freestyle Cells (Invitrogen) using polyethylenimine (PEI). The antibodies were then tested for binding to human GM-CSF by ELISA. Binding properties and V-gene usage is shown in Table 5. The antibodies showed high affinities comparable to the antibodies MOR103 and namilumab, which are GM-CSF neutralizing monoclonal antibodies under clinical development, which serve herein as reference antibodies. The EC50 values (ng/ml) were determined by ELISA and calculated for every sample by nonlinear regression analysis using GraphPad Prism 5 software. The EC50 values ranged from 61.4 to 307.6 ng/ml. Interestingly, the antibodies did not cross-react with both mouse and rat GM-CSF.

The kinetics of binding was determined by surface plasmon resonance (SPR). Briefly, for SPR Protein A (450 nM) was stabilized in 10 mM acetate buffer, pH 4.5, and immobilized onto a EDC/NHS pre-activated ProteOn sensor chip (Biorad) through amine coupling; unreacted groups were blocked by injection of ethanolamine HCl (1 M). HEPES buffered saline (HBS) (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant Tween-20) was used as running buffer. All injections were made at flow rate of 100 □l/min. Monoclonal antibodies were diluted in HBS (200 nM) and injected onto the protein A coated chip for capturing, followed by injection of different concentrations of human GM-CSF (400 nM, 200 nM, 100 nM, 50 nM, 25 nM); one channel of the chip was injected with HBS and used as reference for the analysis. Injection time and dissociation time were 120 s and 600 s, respectively. Each binding interaction of mAbs with GM-CSF was assessed using a ProteON XPR36 instrument (Biorad) and data processed with ProteOn Manager Software. Ka, Kd and KD were calculated applying the Langmuir fit model. The KD determined ranged from 0.18 to 0.69 nM, consistent with the high affinity binding. However, the kinetic values were highly heterogeneous. For instance, antibodies GCA7 and GCB59 had comparable KD values (0.38 and 0.68 nM, respectively), but showed different kinetics with GCA7 being characterized by a slow on-/slow off-rate and GCB59 by a high on-/high off-rate (Table 6).

TABLE 6 Binding properties and V-gene usage of GM-CSF autoantibodies from PAP patients. Human monoclonal antibodies from PAP patients show high affinities comparable to reference monoclonal antibodies. The table shows EC50 values determined by ELISA and Ka, Kd and KD values determined by SPR; reference monoclonal antibodies are highlighted in bold. mAb EC50 (ng/ml) Ka (1/Ms) Kd (1/s) KD (M) GCA7 186.8 2.4E+05 6.0E−05 3.8E−10 GCA21 59.4 9.5E+05 6.5E−04 6.9E−10 GCB59 307.6 1.7E+06 1.2E−03 6.8E−10 GCE536 61.4 6.6E+05 1.1E−04 1.8E−10 Clone 3092 61.0 5.9E+05 3.4E−04 5.7E−10 Clone 1089 1080.0 1.8E+05 7.9E−05 4.4E−10 MOR103 90.0 2.7E+05 1.5E−05 1.9E−10 Namilumab 75.3 3.1E+05 7.7E−05 2.4E−10

Example 2: The Four PAP Autoantibodies Recognize Distinct Sites on GM-CSF and can Form High-Molecular-Weight Immune Complexes

To assess simultaneous binding of the different mABs to GM-CSF, SPR cross competition experiments were performed by SPR as described above (Example 1) whereby the different mAbs (200 nM each) were serially injected after GM-CSF capture (50 nM). Injection time and dissociation time were 60 s and 20 s, respectively. Thereby, it was found that GCA7, GCA21, GCB59 and GCE536 do not cross-compete between them for binding to GM-CSF (FIG. 1A). Interestingly, SPR experiments show that three non-cross-competing autoantibodies can bind simultaneously to a single molecule of GM-CSF (FIG. 1B). Furthermore, when GM-CSF was incubated with an excess of three antibodies, formation of high-molecular-weight immune complexes could be detected by size-exclusion chromatography (SEC-HPLC) (FIG. 1C). In the SEC-HPLC experiment three non-cross competing mAbs were diluted in PBS singularly or as a three-antibody-combination (10 □g of total antibody amount), and mixed with GM-CSF (1:1 or 10:1 molar ratios) for 1 hour, RT. Samples were analyzed by Agilent 1100 HPLC machine using TSK-GEL G3000SW columns (Tosoh, bed volume: 13 ml, void volume: 4.6 ml) with PBS as mobile phase (flow rate: 1 ml/min). A universal solvent 2 □m filter (Agilent) was put between injector and column. Detection was performed by a Variable Wavelength Detector (VWD, Agilent) with ultraviolet absorption at 220 nm.

Example 3: Potent In Vitro Neutralization of GM-CSF Requires Combinations of 3 Antibodies Binding to Non-Overlapping Sites

The neutralizing activity of the autoantibodies was assessed by measuring their ability to inhibit the proliferation of TF-1 cells in response to recombinant GM-CSF (FIG. 2A). To this end, TF-1 cells (CLS, Cell Lines Service) were maintained in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (Hyclone), 1% GlutaMAX, 1% Penicillin/Streptavidin, 1% non-essential amino acids, 1% sodium pyruvate, 1‰ 2-mercaptoethanol (all from GIBCO), 5 ng/ml human GM-CSF (Gentaur), 10 ng/ml human IL-3 (ImmunoTools). Cells were grown at 37° C. in a humidified incubator with 5% CO2. A GM-CSF neutralization assay was performed by serially diluting mAbs (or combination of mAbs, total IgG, or affinity-purified antibodies) in growth medium with neither GM-CSF nor IL-3, adding GM-CSF at a concentration of 100 pg/ml, and preincubating in 96-well flat-bottom cell culture plates (Costar) at 37° C. for 1 hour. TF-1 cells were washed 5 times, diluted in growth medium with neither GM-CSF nor IL-3, and 10,000 cells per well were seeded (final GM-CSF concentration equal to 50 pg/ml). In other tests, GM-CSF was used at final concentration of 500 and 5,000 pg/ml, and 1,000 cells per wells were seeded. Cells with or without GM-CSF in absence of antibodies were used as control to determine maximum and minimum levels of cell proliferation. Plates were incubated at 37° C. in a humidified incubator with 5% CO2 for 72 hours, and cell proliferation was measured after 6-hour incubation with 0.2 □Ci/well of [3H]-thymidine (PerkinElmer). GM-CSF neutralization was calculated as percentage of inhibition of TF-1 growth with the following formula: [1-(CCPM of a single well—average CCPM of control cells grown without GM-CSF)/(average CCPM of control cells grown with GM-CSF−average CCPM of control cells grown without GM-CSF)]×100 (CCPM=corrected counts per minute). IC₉₀ (□g/ml) was calculated for every sample by a nonlinear regression analysis using GraphPad Prism 5 software. In some experiments mouse sera were titrated in TF-1 growth medium and preincubated at 37° C. for 30 min. TF-1 cells were washed and seeded (1,000 cells per well). A titration of GM-CSF (60,000 to 0.3 ng/ml) was added as growth control. CCPM of each single well were plotted against the serum titration.

Surprisingly, using this bioassay, GCA21, GCA7 and GCB59 failed to neutralize GM-CSF (FIG. 2B), even when tested at the concentration of 1 mg/ml (data not shown). The only exception was GCE536 which neutralized GM-CSF activity with an IC₉₀ value of 2.43 μg/ml, while the therapeutic antibodies Namilumab and MOR103 showed IC₉₀ values of 0.80 and 0.16, respectively. Interestingly, when combined together, two non-cross-competing antibodies showed enhanced neutralizing activity both in terms of dose-response and percent inhibition, the combination of GCA21 and GCB59 being the most effective (FIG. 2B). Strikingly, a combination of three non-cross-competing antibodies (GCA21, GCA7 and GCB59) led to a complete inhibition of proliferation with an IC₉₀ value of 0.08 μg/ml (expressed as the total concentration of the three mAbs), which was lower than that of the therapeutic antibodies MOR103 and Namilumab (FIG. 2B).

As expected from the law of mass action, it was found that by varying the cell number and the GM-CSF concentration the sensitivity of the assay was dramatically affected. In particular, lowering the number of TF-1 cells and the concentration of GM-CSF led to a more sensitive test that showed increased neutralization by single and multiple antibodies (FIG. 2C). In contrast, when high number of TF-1 cells and high doses of GM-CSF were used, even the most potent neutralizing antibodies MOR103 and Namilumab, failed to neutralize GM-CSF, even when present in a 400-fold molar excess. Strikingly, in all conditions, a combination of three non-cross-competing antibodies was capable of completely neutralizing GM-CSF (FIG. 2C).

Example 4: FcR-Dependent Clearance of GM-CSF Immune Complexes In Vivo

Having established that GM-CSF can form complexes with three antibodies resulting in efficient in vitro neutralization of the cytokine biological activity, the effect of single versus multiple autoantibodies in vivo was investigated. To this end, groups of 6-8 week-old female BALB/c mice were injected intravenously with 100 □g of purified mAbs or 2 mg of total IgG purified from PA96 patient. After 16 hours, 2 □g of human GM-CSF were injected. Sera samples were collected on day 1 and day 5. GM-CSF was quantified by a sandwich ELISA. Briefly, 10 □g/ml of an antibody that bound to site II of GM-CSF was used to coat 96-well Maxisorp plates (Nunc), which were then blocked with PBS+10% FBS (Gibco). All sera and GM-CSF, which was used as standard (range 3.4-600,000 pg/ml), were titrated and tested in parallel under different conditions (either untreated or after alkaline treatment to dissociate the immune complexes; FIG. 3A): in one plate all samples were supplemented with 25% (vol/vol) of an alkaline dissociation buffer (2.5% Triton X100, 2 M ethanolamine, 0.15 M NaCl, pH 11.6), in the other plate all samples were supplemented with 25% (vol/vol) of PBS+10% FBS. Plates were left overnight at RT. Detection of captured GM-CSF was made with 1 □g/ml of a biotinylated antibody that bound to site I of GM-CSF for 1 h, RT, followed by binding of 0.5 □g/ml streptavidin-AP (Jackson ImmunoResearch) for 1 h, RT. Plates were then washed, substrate (p-NPP, Sigma) was added and plates were read at 405 nm.

In the absence of antibodies, the injected GM-CSF disappeared rapidly from the serum and was undetectable after 24 hours (FIG. 3B). In contrast, when single antibodies (GCA21 or MOR103) were used, high levels of GM-CSF were recovered from serum on day 1 and were still present on day 5. Of note, GM-CSF detection required alkaline dissociation in the case of MOR103 but not for GCA21, consistent with the different dissociation rates of the two antibodies (Table 6). In striking contrast, when mice received three non-cross-competing antibodies (GCA21, GCA7 and GCB59) or PAP IgG, GM-CSF was rapidly cleared since only low or undetectable amounts of the cytokine could be detected in the day-1 and day-5 sera, respectively, after alkaline dissociation.

To address the possible role of Fc receptors in the clearance of GM-CSF, the same antibodies were tested in a variant form, called LALA, which does not bind to C1q or to Fc-γ receptors. Similarly to the wild-type antibodies, single LALA antibodies led to an increase in GM-CSF levels in serum. However, in contrast to what was observed for three wild-type antibodies, three LALA antibodies failed to clear GM-CSF, which was quantitatively recovered in the sera following alkaline dissociation even on day 5 (FIG. 3B).

To ask whether the antibody-bound GM-CSF would be bioavailable, the sera of mice were tested for their ability to support TF-1 proliferation (FIG. 3C). Sera of mice receiving GCA21 or MOR103 led to a robust proliferation of TF-1 cells, consistent with a GM-CSF dissociation rate sufficient to engage the cytokine receptor. In contrast, sera of mice receiving three wild-type antibodies or PAP IgG were not able to stimulate proliferation, consistent with clearance of the immune complexes in vivo. In addition, although containing high level of GM-CSF, sera of mice receiving three LALA antibodies were not stimulatory, a finding consistent with irreversible sequestration of GM-CSF in stable immune complexes.

To further address the role of Fcγ-receptors, immune complexes formed between GM-CSF and wild-type or LALA antibodies were tested for their capacity to bind to TZM-b1 cells expressing different Fcγ-receptors. To this end, four TZM-b1 cell lines (NIH AIDS Research & Reference Reagent Program) each transfected with a specific Fcγ receptor (FcγRI, FcγRIIa, FcγRIIb or FcγRIIIa) were maintained in DMEM medium supplemented with 10% Fetal Bovine Serum (Hyclone), 0.025 M Hepes, 10 □g/ml Gentamicin and 20 □g/ml Blasticidin. Untransfected TZM-b1 cells were used as negative control and were maintained in DMEM medium supplemented with 10% Fetal Bovine Serum (Hyclone) and 2% Penicillin/Streptavidin. Cells were grown at 37° C. in a humidified incubator with 5% CO2. Expression of specific FcγRs was assessed by staining TZM-b1 cells with FITC-conjugated anti-CD64 (anti-FcγRI), anti-CD32 (anti-FcγRIIa and anti-FcγRIIb) and anti-CD16 (anti-FcγRIIIa) antibodies (all from BD Pharmingen). Untrasfected and transfected TZM-b1 cells were washed with staining buffer (PBS with 10% Fetal Bovine Serum and 2 mM EDTA) and seeded in 96-well-plates at a density of 50,000 cells per well. A single anti-GM-CSF mAb (GCA21) or a combination of three non-cross-competing mAbs (GCA21, GCA7 and GCB59) at final concentration of 2.5 □g/ml were mixed with 0.05 □g/ml GM-CSF, or staining buffer (PBS with 10% Fetal Bovine Serum and 2 mM EDTA). The LALA versions of all antibodies and a mAb with a different specificity were included as controls. Samples were incubated at 37° C. for 30 min to allow the formation of immune complexes and then cooled down to 4° C. before adding them to TZM-b1 cells for 30 min. Cells were washed twice and stained with anti-human IgG Fcγ fragment specific F(ab′)2 fragment (Jackson ImmunoResearch). Samples were analyzed on BD FACSCanto (BD Biosciences) and median intensity fluorescence was analyzed and compared between samples.

Strong binding was observed only on FcγRIIa- and FcγRIIb-expressing cells and when immune complexes were formed by three wild-type, but not LALA, antibodies (FIG. 3D). Taken together, the above results indicate that single antibodies, even when potently neutralizing in vitro, increase the half-life of GM-CSF and build up a circulating pool of bioavailable cytokine. In contrast, three or more antibodies lead to the formation of immune complexes that are efficiently cleared through an Fc-dependent mechanism.

Example 5: Engineering of Multispecific Antibodies with the Highest GM-CSF Neutralizing Activity

In view of the need of targeting multiple independent sites on GM-CSF to achieve efficient in vivo neutralization and clearance of the cytokine, bispecific and trispecific antibodies carrying the GCA21, GCA7, GCB59 and GCE536 epitope binding sites, specific linkers and an intact human IgG1 Fc were designed and produced (Table 7). Six different construct types were used to produce the multispecific antibodies (FIG. 4). In particular the three construct types Ts1, Ts2 and Ts3 were used to produce 6 different trispecific hexavalent antibodies, while the three construct types Bs1, Bs2 and Bs3 were used to produce 10 different bispecific tetravalent antibodies (FIG. 4 and Table 7). The construct types Bs1, Bs2, Bs3, Ts1 and Ts2 were designed according to US 2009/0155275 A1.

TABLE 7 Description of GM-CSF multispecific antibodies Ts1GC1 trispecific antibody Construct type Ts1 Specificities¹ GCA21 + GCA7 + GCB59 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCA21 VH 63 65 chain² IgG1 CH1—CH2—CH3 140 146 Short linker 143 149 GCA7 VH 37 42 Long linker 144 150 GCA7 VL 38 47 Short linker 143 149 GCB59 VH 95 100 Long linker 144 150 GCB59 VL 96 104 Complete sequence 151 152 Light GCA21 VL 64 66 chain² IgG CK 141 147 Complete sequence 183 184 Ts1GC2a trispecific antibody Construct type Ts1 Specificities¹ GCE536 + GCA7 + GCB59 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCE536 VH 130 132 chain² IgG1 CH1—CH2—CH3 140 146 Short linker 143 149 GCA7 VH 37 42 Long linker 144 150 GCA7 VL 38 47 Short linker 143 149 GCB59 VH 95 100 Long linker 144 150 GCB59 VL 96 104 Complete sequence 153 154 Light GCE536 VL 131 136 chain² IgG CK 141 147 Complete sequence 189 190 Ts2GC2b trispecific antibody Construct type Ts2 Specificities¹ GCE536 + GCA7 + GCB59 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCB59 VH 95 98 chain² Long linker 144 150 GCB59 VL 96 102 Short linker 143 149 GCE536 VH 130 133 IgG1 CH1—CH2—CH3 140 145 Complete sequence 155 156 Light GCA7 VH 37 41 chain² Long linker 144 150 GCA7 VL 38 46 Short linker 143 149 GCE536 VL 131 137 IgG CK 141 147 Complete sequence 157 158 Ts2GC2c trispecific antibody Construct type Ts2 Specificities¹ GCE536 + GCA7 + GCB59 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCB59 VH 95 99 chain² Long linker 144 150 GCB59 VL 96 103 Short linker 143 149 GCA7 VH 37 40 IgG1 CH1—CH2—CH3 140 145 Complete sequence 159 160 Light GCE536 VH 130 134 chain² Long linker 144 150 GCE536 VL 131 138 Short linker 143 149 GCA7 VL 38 45 IgG CK 141 147 Complete sequence 161 162 Ts3GC2d trispecific antibody Construct type Ts3 Specificities¹ GCE536 + GCA7 + GCB59 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCB59 VH 95 98 chain² Long linker 144 150 GCB59 VL 96 102 Short linker 143 149 GCE536 VH 130 133 IgG1 CH1—CH2—CH3 140 146 Short linker 143 149 GCA7 VH 37 43 Long linker 144 150 GCA7 VL 38 48 Complete sequence 163 164 Light GCE536 VL 131 136 chain² IgG CK 141 147 Complete sequence 189 190 Ts3GC2e trispecific antibody Construct type Ts3 Specificities¹ GCE536 + GCA7 + GCB59 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCB59 VH 95 99 chain² Long linker 144 150 GCB59 VL 96 103 Short linker 143 149 GCA7 VH 37 40 IgG1 CH1—CH2—CH3 140 146 Short linker 143 149 GCE536 VH 130 135 Long linker 144 150 GCE536 VL 131 139 Complete sequence 165 166 Light GCA7 VL 38 44 chain² IgG CK 141 147 Complete sequence 181 182 Bs3GC1a bispecific antibody Construct type Bs3 Specificities¹ GCE536 + GCA7 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCE536 VH 130 132 chain² IgG1 CH1—CH2—CH3 140 146 Short linker 143 149 GCA7 VH 37 43 Long linker 144 150 GCA7 VL 38 48 Complete sequence 167 168 Light GCE536 VL 131 136 chain² IgG CK 141 147 Complete sequence 189 190 Bs3GC1b bispecific antibody Construct type Bs3 Specificities¹ GCE536 + GCA7 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCA7 VH 37 39 chain² IgG1 CH1—CH2—CH3 140 146 Short linker 143 149 GCE536 VH 130 135 Long linker 144 150 GCE536 VL 131 139 Complete sequence 169 170 Light GCA7 VL 38 44 chain² IgG CK 141 147 Complete sequence 181 182 Bs2GC1c bispecific antibody Construct type Bs2 Specificities¹ GCE536 + GCA7 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCE536 VH 130 132 chain² IgG1 CH1—CH2—CH3 140 145 Complete sequence 187 188 Light GCA7 VH 37 41 chain² Long linker 144 150 GCA7 VL 38 46 Short linker 143 149 GCE536 VL 131 137 IgG CK 141 147 Complete sequence 157 158 Bs2GC1d bispecific antibody Construct type Bs2 Specificities¹ GCE536 + GCA7 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCA7 VH 37 39 chain² IgG1 CH1—CH2—CH3 140 145 Complete sequence 179 180 Light GCE536 VH 130 134 chain² Long linker 144 150 GCE536 VL 131 138 Short linker 143 149 GCA7 VL 38 45 IgG CK 141 147 Complete sequence 161 162 Bs1GC2a bispecific antibody Construct type Bs1 Specificities¹ GCE536 + GCB59 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCB59 VH 95 98 chain² Long linker 144 150 GCB59 VL 96 102 Short linker 143 149 GCE536 VH 130 133 IgG1 CH1—CH2—CH3 140 145 Complete sequence 155 156 Light GCE536 VL 131 136 chain² IgG CK 141 147 Complete sequence 189 190 Bs3GC2b bispecific antibody Construct type Bs3 Specificities¹ GCE536 + GCB59 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCB59 VH 95 97 chain² IgG1 CH1—CH2—CH3 140 146 Short linker 143 149 GCE536 VH 130 135 Long linker 144 150 GCE536 VL 131 139 Complete sequence 171 172 Light GCB59 VL 96 101 chain² IgG CL 142 148 Complete sequence 185 186 Bs1GC3a bispecific antibody Construct type Bs1 Specificities¹ GCA7 + GCB59 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCB59 VH 95 99 chain² Long linker 144 150 GCB59 VL 96 103 Short linker 143 149 GCA7 VH 37 40 IgG1 CH1—CH2—CH3 140 145 Complete sequence 159 160 Light GCA7 VL 38 44 chain² IgG CK 141 147 Complete sequence 181 182 Bs3GC3b bispecific antibody Construct type Bs3 Specificities¹ GCA7 + GCB59 Heavy Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl chain² GCB59 VH 95 97 IgG1 CH1—CH2—CH3 140 146 Short linker 143 149 GCA7 VH 37 43 Long linker 144 150 GCA7 VL 38 48 Complete sequence 173 174 Light GCB59 VL 96 101 chain² IgG CL 142 148 Complete sequence 185 186 Bs3GC4 bispecific antibody Construct type Bs3 Specificities¹ GCA21 + GCE536 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCA21 VH 63 65 chain² IgG1 CH1—CH2—CH3 140 146 Short linker 143 149 GCE536 VH 130 135 Long linker 144 150 GCE536 VL 131 139 Complete sequence 175 176 Light GCA21 VL 64 66 chain² IgG CK 141 147 Complete sequence 183 184 Bs3GC5 bispecific antibody Construct type Bs3 Specificities¹ GCA21 + GCA7 Domain/Linker SEQ ID NO. aa SEQ ID NO. nucl Heavy GCA21 VH 63 65 chain² IgG1 CH1—CH2—CH3 140 146 Short linker 143 149 GCA7 VH 37 43 Long linker 144 150 GCA7 VL 38 48 Complete sequence 177 178 Light GCA21 VL 64 66 chain² IgG CK 141 147 Complete sequence 183 184 ¹The antibody used as scaffold is underlined. ²From N-terminus to C-terminus.

Example 6: Evaluation of Productivity and Aggregation of Multispecific Antibodies

The anti GM-CSF multispecific antibodies were produced in 293F cells and purified on protein A. Quantification was performed by Pierce bicinchoninic acid (BCA) protein assay according to the manufacturer's instructions (Thermo Scientific). The assay is a detergent-compatible formulation based on BCA for the colorimetric detection and quantitation of total protein. Productivity varied according to the different antibodies with Bs3GC1a, Bs1GC2a, Bs1GC3a, Bs3GC3b, Bs3GC5 being produced at a concentration greater than 30 μg/ml. Aggregation of multispecific antibodies (final concentration: 1 mg/ml) was analyzed by measuring their turbidity at OD 340 nm in absence or presence of GM-CSF (final concentration: 0.1 mg/ml) (Table 8).

TABLE 8 Productivity (μg/ml) of multispecific mAbs by 293F cells and their aggregation measured by turbidity at OD 340 nm in absence or presence of GM-CSF. Low OD values indicates lower level of turbidity. Turbidity Concentration Turbidity (OD 340) Name (μg/ml) (OD 340) w/GM-CSF Ts1GC1 3.2 0.345 0.369 Ts1GC2a 3 0.292 0.424 Ts2GC2b 7.2 0.076 0.169 Ts2GC2c 7.7 0.068 0.209 Ts3GC2d 13.5 0.012 0.197 Ts3GC2e 11.1 0.108 0.246 Bs3GC1a 30.6 0.084 0.121 Bs3GC1b 17.5 0.051 0.093 Bs2GC1c 7 0.058 0.133 Bs2GC1d 5 0 0.025 Bs1GC2a 41.8 0 0 Bs3GC2b 26 0.044 0.067 Bs1GC3a 61.5 0 0 Bs3GC3b 31.7 0 0 Bs3GC4 20.9 0.553 0.396 Bs3GC5 42 0.224 0.245

Example 7: Multispecific Antibodies Bind to GM-CSF with High Affinity

All multispecific antibodies were tested for binding to GM-CSF by ELISA. The binding was highly specific with a high affinity for GM-CSF as shown by EC50 values in Table 9 below. The use of the different binding sites of the multispecific antibodies was tested by SPR experiments using Ts1GC1 as model. In this experiment GM-CSF was bound by an excess of 2 of the 3 antibodies forming Ts1GC1 and subsequent binding of Ts1GC1 to GM-CSF through the 3rd specificity was revealed. Ts1GC1 has a very high affinity, with very low KD as shown in FIG. 5A. In different SPR experiments GM-CSF was complexed with 2 antibodies out of the 3 antibodies composing Ts1GC1, so that only one of the 3 GM-CSF epitopes was left free for binding by Ts1GC1 (FIG. 5B-D). In addition, when immobilized on the SPR chip, Ts1GC1, unlike conventional antibodies such as GCA7, was able to form high molecular weight complexes when the chip was sequentially exposed to multiple rounds of soluble GM-CSF and Ts1GC1 (FIG. 5E).

TABLE 9 Binding of multispecific mAbs to GM-CSF as determined by ELISA. Name EC50 (ng/ml) Ts1GC1 27.04 Ts1GC2a 33.48 Ts2GC2b 18.38 Ts2GC2c 20.71 Ts3GC2d 29.55 Ts3GC2e 40.67 Bs3GC1a 74.33 Bs3GC1b 71.6 Bs2GC1c 12 Bs2GC1d 22.56 Bs1GC2a 16.19 Bs3GC2b 48.55 Bs1GC3a 46.8 Bs3GC3b 586.7 Bs3GC4 32.27 Bs3GC5 41.75

Example 8: Extremely Potent Neutralization of GM-CSF by Multispecific Antibodies

GM-CSF neutralization was tested using the in vitro bioassay based on TF-1 cells as described above (Example 3), whereby 2 different GM-CSF concentrations (50 and 500 pg/ml) and 2 different number of cells per well (1,000 and 10,000) were tested. IC90 values are reported in Table 10. Interestingly, all multispecific antibodies completely inhibited TF-1 proliferation in all conditions tested at very low concentrations (lower than 1 ng/ml for most multispecific antibodies). Of note, using less stringent conditions MOR103 and Namilumab required a 100-fold and 690-fold greater concentration, respectively, as compared to the best multispecific antibody (Ts1GC2a). Under stringent conditions, most multispecific antibodies could neutralize GM-CSF at concentrations lower than 10 ng/ml, while MOR103 and Namilumab required concentrations greater than 1 mg/ml. Two multispecific antibodies, Ts3GC2d and Bs1GC3a, were selected for their overall properties and compared to the single antibodies or combinations of antibodies from which they derived (FIG. 6).

TABLE 10 Extremely potent neutralization of GM-CSF by multispecific antibodies. Less stringent conditions: 50 pg/ml GM-CSF and 1,000 TF-1/well. Stringent conditions: 50 pg/ml GM- CSF and 10,000 TF-1/well. More stringent conditions: 500 pg/ml GM-CSF and 1,000 TF-1/well. Very stringent conditions: 500 pg/ml GM-CSF and 10,000 TF-1/well IC90 (ng/ml) IC90 (ng/ml) IC90 (ng/ml) less IC90 (ng/ml) more very stringent stringent stringent stringent Name conditions conditions conditions conditions Ts1GC1 0.3516 0.2828 2.965 2.813 Ts1GC2a 0.2649 0.2806 3.173 2.534 Ts2GC2b 0.5257 0.5615 4.705 5.951 Ts2GC2c 0.5465 0.6898 4.284 5.862 Ts3GC2d 0.5363 0.4132 0.7954 0.7681 Ts3GC2e 0.2963 0.2718 3.289 3.684 Bs3GC1a 0.6812 0.9625 4.721 6.235 Bs3GC1b 0.5546 0.7118 4.369 5.928 Bs2GC1c 0.8525 1.239 4.496 6.227 Bs2GC1d 3.075 6.299 9.596 15.89 Bs1GC2a 2.77 31.37 9.917 217.1 Bs3GC2b 0.5595 1.068 6.207 11.18 Bs1GC3a 0.7436 0.7371 4.636 13.3 Bs3GC3b 1.279 2.957 17.81 123.8 Bs3GC4 9.257 90.78 97.5 738.9 Bs3GC5 1.305 4.177 6.465 10.25 MOR103 24.86 139.4 172.3 1490 Namilumab 177.2 798.3 1972 19820

Example 9: Immune Complexes of GM-CSF with Multispecific Antibodies Bind to Fcγ Receptors IIa and IIb

To address the engagement of Fcγ receptors, immune complexes formed between GM-CSF and multispecific antibodies were tested for their capacity to bind to TZM-b1 cells expressing FcγRIIa and FcγRIIb receptors. Multispecific antibodies were mixed with 0.05 μg/ml of GM-CSF, or staining buffer. Samples were incubated at 37° C. for 30 min to allow the formation of immune complexes and then cooled down to 4° C. before adding them to TZM-b1 cells for 30 minutes. Cells were washed twice and stained with anti-human IgG Fcγ fragment specific F(ab′)2 fragment. Samples were analyzed on BD FACSCanto (BD Biosciences) and median intensity fluorescence was analyzed and compared between samples. Strong binding was observed with immune complexes formed by GM-CSF and different multispecific antibodies on FcγRIIa- and FcγRIIb-expressing cells (Table 11). In particular Ts3GC2d and Bs1GC3a showed the strongest binding to FcγRIIa and FcγRIIb in presence of GM-CSF, while they poorly bound the same FcRs in absence of GM-CSF (FIG. 7). Taken together, the above results suggest that GM-CSF in immune complexes with a multispecific monoclonal antibody can be cleared from the human body through an Fc-dependent mechanism.

TABLE 11 Binding of immune complexes formed by GM-CSF and different multispecific mAbs to TZM-bl cells expressing FcγRIIa or FcγRIIb, as measured by flow cytometry using an anti-IgG Fc specific antibody. Shown is the median fluorescence intensity (MFI) of multispecific antibodies alone or complexed with low (1:10 mAb:GM-CSF ratio) or high GM-CSF concentrations (1:1 mAb:GM-CSF ratio), and the average ratio of binding in presence and in absence of GM-CSF. Dark grey cells indicate the GM-CSF complexes formed with multispecific antibodies that poorly bind to FcRs in absence of GM-CSF. FcgRIIa FcgRIIa FcgRIIa FcgRIIb FcgRIIb FcgRIIb (MFI) (MFI) (MFI) Average (MFI) (MFI) (MFI) Average w/o w/ low w/ high RATIO w/o w/ low w/ high RATIO Name GM-CSF GM-CSF GM-CSF FcgRIIa GM-CSF GM-CSF GM-CSF FcgRIIb Ts1GC1 52422 117643 215686 3.18 350077 527988 1230711 2.51 Ts1GC2a 2850 4131 9872 2.46 67215 135509 397219 3.96 Ts2GC2b 11341 135974 725556 37.98 168007 852217 1262147 6.29 Ts2GC2c 9989 91425 189657 14.07 379395 896907 712072 2.12 Ts3GC2d 1313 7898 122101 49.50 28963 343854 651881 17.19 Ts3GC2e 1869 2181 189634 51.31 52524 109361 1236720 12.81 Bs3GC1a 687 1270 65292 48.44 20590 175426 510410 16.65 Bs3GC1b 3548 2937 37183 5.65 53830 60053 378584 4.07 Bs2GC1c 1590 5003 427407 135.98 107869 416283 942605 6.30 Bs2GC1d 2906 10922 319374 56.83 125493 435244 1219560 6.59 Bs1GC2a 662 9141 566165 434.52 11787 398084 1437205 77.85 Bs3GC2b 6906 7127 268634 19.97 96743 181252 1303031 7.67 Bs1GC3a 694 14400 128441 102.91 18221 98121 752715 23.35 Bs3GC3b 650 8290 54962 48.66 2627 57837 432575 93.34 Bs3GC4 6733 24478 19481 3.26 122923 137194 62612 0.81 Bs3GC5 1105 30163 468183 225.50 176650 393019 1578476 5.58

Table of Sequences and SEQ ID Numbers SEQ ID NO Description Sequence GCA7 ANTIBODY   1 CDRH1 aa gftvstny   2 CDRH2 aa l y a gg v t   3 CDRH3 aa akhy d sgystidhfd s   4 CDRL1 aa qsv f y t s k nkny   5 CDRL2 aa was   6 CDRL2 long aa LIYwasTRE   7 CDRL3 aa qqyystpft   8 CDRH1 nuc varS1 GGATTCACCGTCAGTACCAACTAC   9 CDRH1 nuc varS2 GGGTTTACTGTGTCTACAAACTAC  10 CDRH1 nuc varN1 GGCTTTACTGTCTCTACAAACTAC  11 CDRH1 nuc varC1 GGCTTCACCGTGTCAACAAACTAC  12 CDRH1 nuc varC2 GGGTTTACCGTCTCTACAAACTAC  13 CDRH2 nuc varS1 CTTTATGCCGGAGGTGTCACA  14 CDRH2 nuc CTGTACGCTGGCGGGGTGACC varS2/N1/C2  15 CDRH2 nuc varC1 CTGTACGCCGGAGGCGTGACT  16 CDRH3 nuc varS1 GCGAAACACTATGATTCGGGATATTCTACCATAGAT CACTTTGACTCC  17 CDRH3 nuc varS2 GCCAAACACTATGATAGTGGGTACTCCACTATTGAC CATTTTGACTCT  18 CDRH3 nuc varN1 GCCAAACACTATGATAGTGGGTATAGCACAATCGAC CATTTTGACAGC  19 CDRH3 nuc varC1 GCAAAACACTACGATTCTGGGTATAGTACAATTGAC CATTTTGATTCT  20 CDRH3 nuc varC2 GCCAAACACTATGATAGTGGGTACAGTACCATTGAC CATTTCGATAGC  21 CDRL1 nuc varS1 CAGAGTGTTTTCTACACCTCCAAAAATAAAAACTAC  22 CDRL1 nuc varS2 CAGTCCGTCTTCTACACCAGTAAGAACAAAAACTAT  23 CDRL1 nuc varN1 CAGAGCGTGTTCTACACCAGTAAGAACAAAAACTAT  24 CDRL1 nuc varC1 CAGTCCGTGTTCTACACTTCTAAGAACAAAAACTAT  25 CDRL1 nuc varC2 CAGAGTGTCTTCTACACCAGTAAGAACAAAAACTAT  26 CDRL2 nuc varS1 TGGGCATCT  27 CDRL2 nuc TGGGCTAGC varS2/N1/C2  28 CDRL2 nuc varC1 TGGGCCTCA  29 CDRL2 long nuc CTCATTTACTGGGCATCTACCCGGGAG varS1  30 CDRL2 long nuc CTGATCTACTGGGCTAGCACTAGAGAG varS2/N1/C2  31 CDRL2 long nuc CTGATCTACTGGGCCTCAACCCGAGAG varC1  32 CDRL3 nuc varS1 CAGCAATATTATAGTACCCCTTTCACT  33 CDRL3 nuc varS2 CAGCAGTATTATTCTACCCCCTTCACA  34 CDRL3 nuc varN1 CAGCAGTATTACAGCACCCCATTCACA  35 CDRL3 nuc varC1 CAGCAGTACTATAGCACTCCATTCACC  36 CDRL3 nuc varC2 CAGCAGTATTATTCAACACCCTTCACA  37 heavy chain GVQLVQSGGGLVQPGGSLRLSCAASgftvs t nyMSWVRQAP variable domain GKGLEWVSIl y a gg v t RYADSVKTRFTISRDNSKNTLFLQM (VH) aa NALSAEDTAIYYCakhy d sgystidhfd s WGQGTLVTVSS  38 light chain variable DIQMTQSPDSVAVSLGERATINCKSSqsv f y t s k nknyLAWFQ domain (VL) aa QKPGQPPKLLIYwasTRESGVPDRFSGSGSGTDFTLTISSL RPEDVAVYYCqqyystpftFGPGTKVDIK  39 heavy chain ggggtgcaactggtgcagtctgggggaggcttggtccagccgggggggtccctgaga variable domain ctctcctgtgcagcctctGGATTCACCGTCAGTACCAACTACatga (VH) nuc varS1 gctgggtccgccaggctccagggaaggggctggagtgggtctcaattCTTTAT GCCGGAGGTGTCACAaggtacgcagactccgtgaagaccagattcaccat ctccagagacaattccaagaacactctctttcttcaaatgaacgccctgagcgccgaggac acggctatatattactgtGCGAAACACTATGATTCGGGATATTC TACCATAGATCACTTTGACTCCtggggccagggaaccctggtcacc gtctcctca  40 heavy chain GGCGTGCAGCTGGTGCAGAGCGGCGGCGGCCTGGTG variable domain CAGCCTGGAGGGTCACTGAGACTGTCATGCGCAGCAA (VH) nuc varS2 GCGGGTTTACTGTGTCTACAAACTACATGTCTTGGG TGAGGCAGGCACCTGGAAAGGGACTGGAGTGGGTCTC AATCCTGTACGCTGGCGGGGTGACCCGGTATGCAGA CAGCGTCAAGACCCGGTTCACAATTAGCAGAGATAACT CCAAAAATACTCTGTTTCTGCAGATGAATGCCCTGTCC GCTGAAGACACCGCAATCTACTATTGCGCCAAACACTA TGATAGTGGGTACTCCACTATTGACCATTTTGACTC TTGGGGGCAGGGGACTCTGGTGACTGTCTCTTCA  41 heavy chain GGCGTCCAGCTGGTGCAGAGCGGAGGGGGCCTGGTG variable domain CAGCCTGGCGGGTCCCTGAGACTGAGTTGTGCCGCAA (VH) nuc varN1 GTGGCTTTACTGTCTCTACAAACTACATGTCTTGGG TGAGGCAGGCACCTGGAAAGGGACTGGAGTGGGTCTC AATCCTGTACGCTGGCGGGGTGACCCGGTATGCAGA CAGCGTCAAGACCCGGTTCACAATTAGCAGAGATAACT CCAAAAATACTCTGTTTCTGCAGATGAATGCCCTGTCC GCTGAAGACACCGCAATCTACTATTGCGCCAAACACTA TGATAGTGGGTATAGCACAATCGACCATTTTGACAG CTGGGGACAGGGAACTCTGGTGACAGTCTCATCA  42 heavy chain GGAGTGCAGCTGGTCCAGAGCGGAGGAGGACTGGTG variable domain CAGCCAGGAGGGTCACTGAGGCTGAGCTGCGCAGCTT (VH) nuc varC1 CCGGCTTCACCGTGTCAACAAACTACATGAGCTGGGT CCGCCAGGCACCTGGGAAGGGACTGGAGTGGGTGTCC ATCCTGTACGCCGGAGGCGTGACTCGATATGCTGAC TCTGTCAAGACTCGGTTCACCATCTCTAGAGATAACAG TAAGAACACCCTGTTTCTGCAGATGAATGCACTGAGT GCCGAAGACACAGCTATCTACTATTGTGCAAAACACTA CGATTCTGGGTATAGTACAATTGACCATTTTGATTC TTGGGGCCAGGGGACACTGGTGACTGTCAGCTCC  43 heavy chain GGCGTGCAGCTGGTCCAGAGCGGAGGCGGACTGGTCC variable domain AGCCCGGCGGATCACTGAGACTGTCATGTGCCGCAAG (VH) nuc varC2 CGGGTTTACCGTCTCTACAAACTACATGTCTTGGGT GAGGCAGGCACCTGGAAAGGGACTGGAGTGGGTCTCA ATCCTGTACGCTGGCGGGGTGACCCGGTATGCAGAC AGCGTCAAGACCCGGTTCACAATTAGCAGAGATAACTC CAAAAATACTCTGTTTCTGCAGATGAATGCCCTGTCCG CTGAAGACACCGCAATCTACTATTGCGCCAAACACTAT GATAGTGGGTACAGTACCATTGACCATTTCGATAGC TGGGGGCAGGGGACTCTGGTGACCGTCTCATCA  44 light chain variable gacatccagatgacccagtctccagactccgtggctgtgtctctgggcgagagggccacc domain (VL) nuc atcaactgcaagtccagcCAGAGTGTTTTCTACACCTCCAAAAA varS1 TAAAAACTACttagcttggttccagcagaaaccaggacagcctcctaaactgctc atttacTGGGCATCTacccgggagtccggggtccctgaccgattcagtggcag cgggtctgggacagatttcactctcaccatcagcagcctgcggcctgaagatgtggcagt ttattactgtCAGCAATATTATAGTACCCCTTTCACTttcggccc tgggaccaaagtggatatcaaa  45 light chain variable GACATTCAGATGACCCAGAGTCCTGACAGCGTGGCCG domain (VL) nuc TCTCACTGGGGGAAAGGGCTACTATCAATTGTAAAAG varS2 TTCACAGTCCGTCTTCTACACCAGTAAGAACAAAAAC TATCTGGCCTGGTTTCAGCAGAAGCCAGGCCAGCCCCC TAAACTGCTGATCTACTGGGCTAGCACTAGAGAGTCT GGAGTGCCAGACAGATTCTCTGGCAGTGGGTCAGGAA CCGACTTCACCCTGACAATTAGCTCCCTGAGGCCCGAA GACGTGGCCGTCTATTATTGTCAGCAGTATTATTCTA CCCCCTTCACATTCGGACCTGGGACTAAAGTGGATAT CAAA  46 light chain variable GACATTCAGATGACCCAGAGTCCTGATTCCGTGGCTG domain (VL) nuc TCTCACTGGGGGAGCGAGCAACTATTAACTGCAAGTC varN1 TTCACAGAGCGTGTTCTACACCAGTAAGAACAAAAAC TATCTGGCCTGGTTTCAGCAGAAGCCAGGCCAGCCCCC TAAACTGCTGATCTACTGGGCTAGCACTAGAGAGTCT GGAGTGCCAGACAGATTCTCTGGCAGTGGGTCAGGAA CCGACTTCACCCTGACAATTAGCTCCCTGAGGCCCGAA GACGTGGCCGTCTACTATTGTCAGCAGTATTACAGCA CCCCATTCACATTCGGCCCTGGAACCAAAGTGGATATT AAG  47 light chain variable GACATCCAGATGACTCAGTCTCCCGATAGTGTGGCCG domain (VL) nuc TCTCCCTGGGGGAGAGGGCTACAATTAACTGCAAGAG varC1 CTCCCAGTCCGTGTTCTACACTTCTAAGAACAAAAAC TATCTGGCATGGTTTCAGCAGAAGCCTGGACAGCCCC CTAAACTGCTGATCTACTGGGCCTCAACCCGAGAGAG CGGAGTCCCAGACAGATTCTCAGGCAGCGGGTCCGGA ACAGATTTTACCCTGACAATTTCTAGTCTGCGGCCTGA AGACGTGGCTGTCTACTATTGTCAGCAGTACTATAGC ACTCCATTCACCTTTGGCCCCGGGACAAAGGTGGATA TCAAA 48 light chain variable GATATTCAGATGACCCAGAGTCCTGATTCCGTCGCTG domain (VL) nuc TCTCACTGGGAGAAAGGGCAACCATTAACTGTAAAAG varC2 CTCACAGAGTGTCTTCTACACCAGTAAGAACAAAAAC TATCTGGCCTGGTTTCAGCAGAAGCCAGGCCAGCCCCC TAAACTGCTGATCTACTGGGCTAGCACTAGAGAGTCT GGAGTGCCAGACAGATTCTCTGGCAGTGGGTCAGGAA CCGACTTCACCCTGACAATTAGCTCCCTGAGGCCCGAA GACGTGGCCGTCTACTATTGTCAGCAGTATTATTCAA CACCCTTCACATTCGGACCAGGAACAAAAGTGGATAT TAAG GCA21 ANTIBODY  49 CDRH1 aa gftfs n y p  50 CDRH2 aa i lp dg nr k  51 CDRH3 aa t rdgtyy sn ggvyqtyrr f fd f  52 CDRL1 aa q n i ln w  53 CDRL2 aa kas  54 CDRL2 long aa LIYkas DLQ  55 CDRL3 aa q h ynsy p lt  56 CDRH1 nuc GGATTCACCTTTTCGAACTATCCT  57 CDRH2 nuc ATTTTACCTGATGGGAACAGAAAA  58 CDRH3 nuc ACGAGAGATGGCACGTATTACTCTAATGGTGGTGT TTATCAGACATATCGAAGGTTCTTCGATTTC  59 CDRL1 nuc CAGAATATCCTTAATTGG  60 CDRL2 nuc AAGGCGTCT  61 CDRL2 long nuc ctgatatatAAGGCGTCTgatttacaa  62 CDRL3 nuc CAGCATTATAATAGTTATCCTCTCACT  63 heavy chain QVQLMESGGGVVQPGRSLRLSCSAF gftfs n y p MHWVRQAP variable domain GKGLEWVAI i lp dg nr k NYGRSVTGRFTISRDNSNNSLYLQ (VH) aa MNNLTTEDTAMYYC t rdgtyy sn ggvyqtyrr f fd f WGRGTLVT VSS  64 light chain variable DIQMTQSPSTLSTSVGDRVTITCRASq n i ln wLAWYQQKPG domain (VL) aa NAPNLLIYkas DLQSGVPSRFSGSGSGTEFTLTISSLQPDDF ATYYCq h ynsy p ltFGGGTKVEIK  65 heavy chain caggtgcaattgatggagtctgggggaggcgtggtccagcctgggaggtccctgcgac variable domain tctcatgcagtgcctttGGATTCACCTTTTCGAACTATCCTatgca (VH) nuc ctgggtccgccaggctccaggcaagggacttgagtgggtggctatcATTTTACC TGATGGGAACAGAAAAaactatggaaggtccgtgacgggccgattcacc atctccagagacaattccaacaacagcctttatttgcaaatgaacaacctgacgactgagga cacggctatgtactattgtACGAGAGATGGCACGTATTACTCTA ATGGTGGTGTTTATCAGACATATCGAAGGTTCTTC GATTTCtggggccgtggcaccctggtcaccgtctcctca  66 light chain variable gacatccagatgacccagtctccttccaccctgtctacatctgtgggagacagagtcaccat domain (VL) nuc cacttgccgggccagtCAGAATATCCTTAATTGGttggcctggtatcaa cagaaaccagggaacgcccctaacctcctgatatatAAGGCGTCTgatttacaaa gtggggtcccctcaagattcagcggcagtgggtctgggacagaattcactctcaccatca gcagcctgcagcctgatgattttgcaacttattactgcCAGCATTATAATAG TTATCCTCTCACTttcggcggagggaccaaggtggaaatcaaa GCB59 ANTIBODY  67 CDRH1 aa G LS FSS SG  68 CDRH2 aa IS G S QN Y K  69 CDRH3 aa VG GFPYWLPPS DF SGFHV  70 CDRL1 aa NIGSKS  71 CDRL2 aa A D N  72 CDRL2 long aa VVY A D N DRP  73 CDRL3 aa QVWD GNT DHVV  74 CDRH1 nuc varS1 GGATTGTCCTTCAGTAGTTCAGGC  75 CDRH1 nuc GGCCTGTCCTTCAGCTCCTCTGGC varN1/N2  76 CDRH1 nuc varC1 GGGCTGAGCTTCAGCTCCTCTGGA  77 CDRH2 nuc varS1 ATTAGTGGTAGTCAGAACTACAAA  78 CDRH2 nuc ATTAGCGGGTCCCAGAATTACAAG varN1/N2  79 CDRH2 nuc varC1 ATTTCTGGCAGTCAGAATTACAAG  80 CDRH3 nuc varS1 GTGGGAGGTTTCCCCTATTGGTTACCCCCGAGCGAC TTCTCCGGTTTCCATGTC  81 CDRH3 nuc GTCGGCGGGTTTCCCTATTGGCTGCCTCCAAGCGAC varN1/N2 TTTTCAGGGTTTCATGTC  82 CDRH3 nuc varC1 GTCGGGGGATTTCCCTATTGGCTGCCCCCTTCCGAT TTCTCTGGCTTTCACGTG  83 CDRL1 nuc varS1 AACATTGGAAGTAAAAGT  84 CDRL1 nuc AACATCGGCAGCAAGAGC varN1/N2  85 CDRL1 nuc varC1 AACATCGGGTCTAAGAGT  86 CDRL2 nuc varS1 GCTGATAAC  87 CDRL2 nuc varN1/ GCTGACAAC N2  88 CDRL2 nuc varC1 GCCGACAAT  89 CDRL2 long nuc GTCGTCTATGCTGATAACGACAGGCCC varS1  90 CDRL2 long nuc GTGGTCTATGCTGACAACGATCGGCCC varN1/N2  91 CDRL2 long nuc GTGGTCTATGCCGACAATGATCGGCCA varC1  92 CDRL3 nuc var S1 CAGGTGTGGGATGGTAATACTGATCATGTGGTC  93 CDRL3 nuc CAGGTCTGGGATGGGAATACTGACCACGTCGTC varN1/N2  94 CDRL3 nuc varC1 CAGGTCTGGGACGGGAACACAGATCATGTGGTC  95 heavy chain EVQLVESGGDLVKAGGSLRLSCAVSG LS FSS SG MNWVRQ variable domain APGKGLEWISSIS G S QN Y K YYADSVKGRFVVSRDNARNFL (VH) aa YLQMDSLRAEDTAVYFC VG GFPYWLPPS DF SGFHVWG QGTTVTVSS  96 light chain variable SYVLTQPPSVSVAPGQTASLTCGGT NIGSKSVHWYQQKA domain (VL) aa GQAPVLVVY A D N DRPSGVPERFSGSNSGNTATLTISRVEA EDESDYFCQVWD GNT DHVVFGGGTKLTVL  97 heavy chain gaggtacaattggtggagtctgggggagacctggtcaaggcgggggggtccctgaga variable domain ctctcctgtgccgtctctggattgtccttcagtagttcaggcatgaattgggtccgccag (VH) nuc varS1 gctccagggaaggggctggagtggatctcatcgattagtggtagtcagaactacaaa tactatgcagactcagtgaagggccgattcgtcgtctccagagacaacgcccgcaactttc tatatctgcaaatggacagcctgagggccgaggatacggctgtgtatttttgtgtggga ggtttcccctattggttacccccgagcgacttctccggtttccatgtctggggccaa gggaccacggtcaccgtctcctca  98 heavy chain GAGGTGCAGCTGGTGGAAAGCGGAGGGGATCTGGTG variable domain AAAGCAGGAGGGAGCCTGAGACTGTCATGCGCCGTGA (VH) nuc var N1 GCGGGCTGTCATTCAGCTCCTCTGGCATGAACTGGG TGCGACAGGCTCCTGGAAAGGGACTGGAGTGGATCAG TTCAATTAGCGGGTCCCAGAATTACAAGTACTATGCA GACTCTGTCAAAGGAAGGTTCGTGGTCAGCCGGGATA ACGCCAGAAATTTTCTGTATCTGCAGATGGACAGCCT GCGCGCCGAAGATACCGCCGTGTACTTCTGCGTCGGC GGGTTTCCCTATTGGCTGCCTCCAAGCGATTTCAGC GGATTTCATGTCTGGGGGCAGGGAACTACAGTGACC GTCTCATCA  99 heavy chain GAGGTGCAGCTGGTGGAAAGTGGGGGCGATCTGGTC variable domain AAAGCCGGAGGGTCTCTGCGACTGTCTTGTGCTGTGA (VH) nuc var N2 GCGGCCTGTCCTTCAGCTCCTCTGGCATGAACTGGG TGCGACAGGCTCCTGGAAAGGGACTGGAGTGGATCAG TTCAATTAGCGGGTCCCAGAATTACAAGTACTATGCA GACTCTGTCAAAGGAAGGTTCGTGGTCAGCCGGGATA ACGCCAGAAATTTTCTGTATCTGCAGATGGACAGCCT GCGCGCCGAAGATACCGCCGTGTACTTCTGCGTCGGC GGGTTTCCCTATTGGCTGCCTCCAAGCGACTTTTCA GGGTTTCATGTCTGGGGGCAGGGAACTACCGTGACC GTCTCATCT 100 heavy chain GAGGTGCAGCTGGTCGAATCTGGCGGGGACCTGGTG variable domain AAGGCAGGAGGCAGTCTGAGGCTGTCATGCGCCGTCT (VH) nuc varC1 CAGGGCTGAGCTTCAGCTCCTCTGGAATGAACTGGG TGCGCCAGGCACCAGGCAAAGGACTGGAGTGGATCAG TTCAATTTCTGGCAGTCAGAATTACAAGTACTATGCT GACAGTGTGAAAGGGCGATTCGTGGTCTCCCGGGATA ACGCAAGAAATTTTCTGTATCTGCAGATGGACAGCCT GAGAGCCGAAGATACTGCTGTGTACTTCTGTGTCGG GGGATTTCCCTATTGGCTGCCCCCTTCCGATTTCTC TGGCTTTCACGTGTGGGGACAGGGCACCACAGTGACC GTCAGCTCC 101 light chain variable TCATATGTGCTGACTCAACCACCCTCGGTGTCAGTGGC domain (VL) nuc CCCAGGACAGACGGCCAGTCTAACCTGTGGGGGAACT varS1 AACATTGGAAGTAAAAGTGTTCATTGGTACCAGCAAA AGGCAGGCCAGGCCCCTGTGTTGGTCGTCTATGCTGA TAACGACAGGCCCTCAGGGGTCCCTGAGCGATTCTCT GGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAG CAGGGTCGAGGCCGAGGATGAGTCCGACTATTTCTGT CAGGTGTGGGATGGTAATACTGATCATGTGGTCTT CGGCGGAGGGACCAAGCTGACCGTCCTG 102 light chain variable TCTTACGTCCTGACCCAGCCACCTAGCGTGAGCGTCGC domain (VL) nuc ACCAGGGCAGACAGCTTCACTGACTTGCGGAGGCACA var N1 AACATTGGCAGCAAGAGCGTGCACTGGTACCAGCAGA AAGCCGGACAGGCTCCCGTCCTGGTGGTCTATGCTGA CAACGATCGGCCCTCTGGCGTGCCTGAAAGATTCAGC GGCTCCAACTCTGGGAATACCGCAACACTGACCATCAG TAGGGTCGAGGCCGAAGACGAGTCAGATTACTTTTGC CAGGTGTGGGACGGCAATACTGACCATGTCGTGTTC GGCGGCGGGACCAAACTGACTGTGCTG 103 light chain variable TCCTACGTCCTGACTCAGCCACCTAGCGTGTCCGTCGC domain (VL) nuc ACCTGGGCAGACAGCATCACTGACTTGCGGGGGAACC var N2 AACATCGGCAGCAAGAGCGTGCACTGGTACCAGCAGA AAGCCGGACAGGCTCCCGTCCTGGTGGTCTATGCTGA CAACGATCGGCCCTCTGGCGTGCCTGAAAGATTCAGC GGCTCCAACTCTGGGAATACCGCAACACTGACCATCAG TAGGGTCGAGGCCGAAGACGAGTCAGATTACTTTTGC CAGGTCTGGGATGGGAATACTGACCACGTCGTCTTC GGAGGCGGAACCAAACTGACTGTCCTG 104 light chain variable TCCTACGTGCTGACTCAGCCACCTAGCGTGTCCGTCGC domain (VL) nuc ACCTGGACAGACTGCCAGCCTGACCTGCGGAGGAACAA varC1 ACATCGGGTCTAAGAGTGTGCACTGGTACCAGCAGAA AGCCGGACAGGCTCCCGTCCTGGTGGTCTATGCCGAC AATGATCGGCCATCTGGCGTGCCCGAAAGATTCTCAG GAAGCAACTCCGGCAATACCGCTACACTGACTATTTCT AGGGTGGAGGCAGAAGACGAGAGTGATTATTTCTGT CAGGTCTGGGACGGGAACACAGATCATGTGGTCTTT GGAGGCGGGACCAAGCTGACAGTGCTG GCE536 ANTIBODY 105 CDRH1 aa GY V FTSYY 106 CDRH2 aa I S P GDVN T 107 CDRH3 aa ARGPRSK PP YLYF AL DV 108 CDRL1 aa QSVSSS L 109 CDRL2 aa GAS 110 CDRL2 long aa LIYGAS NRA 111 CDRL3 aa Q H YGSRVT 112 CDRH1 nuc varS1 GGATACGTGTTCACCTCTTACTAT 113 CDRH1 nuc varS2 GGATACGTCTTTACCTCTTACTAT 114 CDRH1 nuc GGATACGTCTTCACCTCTTACTAT varN1/C1 115 CDRH2 nuc ATCTCTCCCGGAGACGTGAACACT varS1/S2/N1/C1 116 CDRH3 nuc varS1 GCTAGGGGGCCCCGCAGCAAGCCTCCTTATCTGTAT TTTGCTCTGGATGTG 117 CDRH3 nuc varS2 GCTAGGGGGCCCCGCAGCAAGCCTCCTTATCTGTAC TTCGCTCTGGATGTC 118 CDRH3 nuc GCTAGGGGGCCCCGCAGCAAGCCTCCTTATCTGTAT varN1/C1 TTCGCTCTGGATGTC 119 CDRL1 nuc varS1 CAGAGTGTCAGCAGCAGCCTC 120 CDRL1 nuc varS2 CAGTCTGTGAGCTCCTCTCTG 121 CDRL1 nuc CAGTCCGTGAGCTCCTCTCTG varN1/C1 122 CDRL2 nuc varS1 GGTGCATCC 123 CDRL2 nuc GGCGCCTCC varS2/N1/C1 124 CDRL2 long nuc CTCATCTACGGTGCATCCAATAGGGCC varS1 125 CDRL2 long nuc CTGATCTATGGCGCCTCCAACCGCGCT varS2/N1/C1 126 CDRL3 nuc varS1 CAGCACTATGGCTCACGGGTCACT 127 CDRL3 nuc varS2 CAGCACTATGGCAGCAGGGTCACT 128 CDRL3 nuc varN1 CAGCATTATGGGTCACGGGTCACT 129 CDRL3 nuc varC1 CAGCATTATGGAAGCAGGGTCACC 130 heavy chain QLQLVQSGTEVKKPGASVKVSCKSSGY V FTSYY LVWVRQ variable domain APGQGLEWMAT I S P GDVN TSYEQRFQGRVTVTTDASTN (VH) aa TVDMELRSLRSEDTAVYYCARGPRSK PP YLYF AL DVWG QGTAVTVSS 131 light chain variable EIVLTQSPGTLSLSPGETAILSCRASQSVSSS L LAWYQQKP domain (VL) aa GQAPRLLIYGAS NRATGIRGRFSGSGSGTDFTLTISRLEPE DFVLYYCQ H YGSRVTFGQGTKLEIK 132 heavy chain CAGCTGCAGCTGGTCCAGTCAGGCACAGAGGTCAAAA variable domain AGCCAGGAGCATCAGTGAAGGTGTCTTGTAAGTCATC (VH) nuc varS1 AGGATACGTGTTCACCTCTTACTATCTGGTGTGGGT CCGGCAGGCACCAGGACAGGGACTGGAGTGGATGGCC ACAATCTCTCCCGGAGACGTGAACACTAGTTACGAAC AGCGATTCCAGGGCAGAGTGACCGTCACCACAGACGCT TCAACTAATACCGTGGATATGGAGCTGCGGAGCCTGA GATCCGAAGATACAGCCGTCTACTATTGCGCTAGGGG GCCCCGCAGCAAGCCTCCTTATCTGTATTTTGCTCT GGATGTGTGGGGGCAGGGGACCGCTGTCACCGTGTC AAGC 133 heavy chain CAGCTGCAGCTGGTCCAGTCAGGCACAGAAGTCAAAAA variable domain ACCCGGCGCAAGCGTGAAGGTCTCATGTAAATCATCA (VH) nuc varS2 GGATACGTCTTTACCTCTTACTATCTGGTGTGGGTC CGGCAGGCACCAGGACAGGGACTGGAGTGGATGGCCA CAATCTCTCCCGGAGACGTGAACACTAGTTACGAACA GCGATTCCAGGGCAGAGTGACCGTCACCACAGACGCT TCAACTAATACCGTGGATATGGAGCTGCGGAGCCTGA GATCCGAAGATACAGCCGTCTACTATTGCGCTAGGGG GCCCCGCAGCAAGCCTCCTTATCTGTACTTCGCTCTG GATGTCTGGGGGCAGGGGACCGCCGTCACCGTCTCAA GC 134 heavy chain CAGCTGCAGCTGGTCCAGAGCGGCACAGAGGTGAAAA variable domain AGCCAGGAGCATCAGTCAAAGTGTCTTGTAAGTCATC (VH) nuc varN1 AGGATACGTCTTCACCTCTTACTATCTGGTGTGGGT CCGGCAGGCACCAGGACAGGGACTGGAGTGGATGGCC ACAATCTCTCCCGGAGACGTGAACACTAGTTACGAAC AGCGATTCCAGGGCAGAGTGACCGTCACCACAGACGCT TCAACTAATACCGTGGATATGGAGCTGCGGAGCCTGA GATCCGAAGATACAGCCGTCTACTATTGCGCTAGGGG GCCCCGCAGCAAGCCTCCTTATCTGTATTTCGCTCT GGATGTCTGGGGGCAGGGAACAGCAGTCACCGTCTCT TCT 135 heavy chain CAGCTGCAGCTGGTCCAGAGCGGAACCGAAGTGAAGA variable domain AACCCGGCGCAAGCGTCAAAGTCTCATGCAAATCAAGC (VH) nuc varC1 GGATACGTCTTCACCTCTTACTATCTGGTGTGGGTC CGGCAGGCACCAGGACAGGGACTGGAGTGGATGGCCA CAATCTCTCCCGGAGACGTGAACACTAGTTACGAACA GCGATTCCAGGGCAGAGTGACCGTCACCACAGACGCT TCAACTAATACCGTGGATATGGAGCTGCGGAGCCTGA GATCCGAAGATACAGCCGTCTACTATTGCGCTAGGGG GCCCCGCAGCAAGCCTCCTTATCTGTATTTCGCTCT GGATGTCTGGGGGCAGGGAACAGCAGTCACCGTCTCA AGC 136 light chain variable gaaattgtgttgacgcagtctcctggcaccctgtctttgtctccaggggaaacagccatcct domain (VL) nuc ctcctgcagggccagtcagagtgtcagcagcagcctcttagcctggtaccagcaaaaa varS1 cctggccaggctcccaggctcctcatctacggtgcatccaatagggccactggcatcaga ggcaggtttagtggcagtgggtctgggacagacttcactctcaccatcagtagattggag cctgaagattttgtactttattactgtcagcactatggctcacgggtcacttttggccag gggaccaagctggagatcaaac 137 light chain variable GAAATCGTGCTGACCCAGTCTCCTGGAACTCTGTCTCT domain (VL) nuc GTCACCTGGCGAAACCGCAATCCTGTCCTGTAGGGCAA varS2 GTCAGTCTGTGAGCTCCTCTCTGCTGGCATGGTACC AGCAGAAGCCCGGACAGGCCCCTAGGCTGCTGATCTA TGGCGCCTCCAACCGCGCTACTGGCATTCGGGGGAGA TTCAGTGGCTCAGGGAGCGGAACCGACTTTACCCTGA CAATCAGCCGGCTGGAGCCCGAAGATTTCGTGCTGTA TTACTGTCAGCACTATGGCAGCAGGGTCACTTTTGG GCAGGGGACTAAACTGGAGATTAAA 138 light chain variable GAAATCGTCCTGACCCAGTCACCTGGCACCCTGAGTCT domain (VL) nuc GAGTCCTGGCGAAACAGCAATCCTGTCTTGTCGGGCT varN1 TCACAGTCCGTGAGCTCCTCTCTGCTGGCATGGTACC AGCAGAAGCCCGGACAGGCCCCTAGGCTGCTGATCTA TGGCGCCTCCAACCGCGCTACTGGCATTCGGGGGAGA TTCAGTGGCTCAGGGAGCGGAACCGACTTTACCCTGA CAATCAGCCGGCTGGAGCCCGAAGATTTCGTGCTGTA CTACTGTCAGCATTATGGGTCACGGGTCACTTTTGG GCAGGGGACTAAACTGGAAATCAAG 139 light chain variable GAGATTGTCCTGACCCAGTCACCTGGCACCCTGAGCCT domain (VL) nuc GAGTCCTGGAGAGACCGCTATTCTGTCTTGTCGGGCA varC1 TCACAGTCCGTGAGCTCCTCTCTGCTGGCATGGTACC AGCAGAAGCCCGGACAGGCCCCTAGGCTGCTGATCTA TGGCGCCTCCAACCGCGCTACTGGCATTCGGGGGAGA TTCAGTGGCTCAGGGAGCGGAACCGACTTTACCCTGA CAATCAGCCGGCTGGAGCCCGAAGATTTCGTGCTGTA CTATTGTCAGCATTATGGAAGCAGGGTCACCTTCGG ACAGGGAACTAAACTGGAAATCAAG Constant regions 140 IgG1 CH1-CH2- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS CH3 aa WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 141 IgG CK aa RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC 142 IgG CL aa GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQW KSHRSYSCQVTHEGSTVEKTVAPTECS 143 Short linker aa GGGGS 144 Long linker aa GGGGSGGGGSGGGGS 145 IgG1 CH1-CH2- gcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctggg CH3 nucl varS1 ggcacagcggccctgggctgcctggtcaaggactacttccccgaacctgtgacggtctc gtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctc aggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagac ctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcc caaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggggga ccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctga ggtcacatgcgtggtggtggacgtgagccacgaGgaTcctgaggtcaagttcaactg gtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagta caacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatgg caaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatc tccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggga ggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcga catcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcct cccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagca ggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccact acacgcagaagagcctctccctgtccccgggtaaa 146 IgG1 CH1-CH2 GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACC CH3 nucl varS2 CTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGG GCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACG GTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT GGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC CCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAA TCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGA GGATCCTGAAGTCAAGTTCAACTGGTACGTGGATGGC GTCGAGGTGCATAATGCCAAGACAAAACCCCGGGAGG AACAGTACAACTCAACTTATAGAGTCGTGAGCGTCCTG ACCGTGCTGCATCAGGACTGGCTGAACGGCAAAGAAT ACAAGTGCAAAGTGTCTAATAAGGCCCTGCCTGCTCCA ATCGAGAAAACAATTAGCAAGGCAAAAGGGCAGCCCAG GGAACCTCAGGTGTACACTCTGCCTCCAAGCCGCGAGG AAATGACCAAGAACCAGGTCTCCCTGACATGTCTGGTG AAAGGATTCTATCCTAGTGACATTGCCGTGGAGTGGG AATCAAATGGCCAGCCAGAGAACAATTACAAGACCACA CCCCCTGTGCTGGACTCTGATGGGAGTTTCTTTCTGT ATTCCAAGCTGACCGTGGATAAATCTAGATGGCAGCA GGGAAATGTCTTTAGCTGTTCCGTGATGCATGAGGCA CTGCACAACCATTACACCCAGAAATCACTGTCACTGTC CCCAGGAAAA 147 IgG CK nucl cgTacGgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctg gaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtg gaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggaca gcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgag aaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaag agcttcaacaggggagagtgt 148 IgG CL nucl ggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaag ccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtgg cttggaaagcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaa caaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaa gtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagaca gtggcccctacagaatgttca 149 Short linker nucl GGCGGGGGAGGCTCT 150 Long linker nucl GGCGGGGGAGGCTCTGGGGGAGGCGGGAGTGGAGGC GGGGGATCA Engineered chains of multtspecific antibodies 151 Ts1GC1 heavy QVQLMESGGGVVQPGRSLRLSCSAFGFTFSNYPMHWVR chain aa QAPGKGLEWVAIILPDGNRKNYGRSVTGRFTISRDNSNN SLYLQMNNLTTEDTAMYYCTRDGTYYSNGGVYQTYRR FFDFWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGVQLV QSGGGLVQPGGSLRLSCAASGFTVSTNYMSWVRQAPGK GLEWVSILYAGGVTRYADSVKTRFTISRDNSKNTLFLQM NALSAEDTAIYYCAKHYDSGYSTIDHFDSWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPDSVAVSLGERATINC KSSQSVFYTSKNKNYLAWFQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLRPEDVAVYYCQQYYSTPF TFGPGTKVDIKGGGGSEVQLVESGGDLVKAGGSLRLSCA VSGLSFSSSGMNWVRQAPGKGLEWISSISGSQNYKYYADS VKGRFVVSRDNARNFLYLQMDSLRAEDTAVYFCVGGFP YWLPPSDFSGFHVWGQGTTVTVSSGGGGSGGGGSGGG GSSYVLTQPPSVSVAPGQTASLTCGGTNIGSKSVHWYQQ KAGQAPVLVVYADNDRPSGVPERFSGSNSGNTATLTISRV EAEDESDYFCQVWDGNTDHVVFGGGTKLTVL 152 Ts1GC1 heavy CAGGTGCAATTGATGGAGTCTGGGGGAGGCGTGGTC chain nucl CAGCCTGGGAGGTCCCTGCGACTCTCATGCAGTGCCT TTGGATTCACCTTTTCGAACTATCCTATGCACTGGGT CCGCCAGGCTCCAGGCAAGGGACTTGAGTGGGTGGCT ATCATTTTACCTGATGGGAACAGAAAAAACTATGGAA GGTCCGTGACGGGCCGATTCACCATCTCCAGAGACAAT TCCAACAACAGCCTTTATTTGCAAATGAACAACCTGAC GACTGAGGACACGGCTATGTACTATTGTACGAGAGAT GGCACGTATTACTCTAATGGTGGTGTTTATCAGACAT ATCGAAGGTTCTTCGATTTCTGGGGCCGTGGCACCCT GGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGG TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGG GGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACT TCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCC CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC AACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA GAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGG TGGACGTGAGCCACGAGGATCCTGAAGTCAAGTTCAA CTGGTACGTGGATGGCGTCGAGGTGCATAATGCCAAG ACAAAACCCCGGGAGGAACAGTACAACTCAACTTATAG AGTCGTGAGCGTCCTGACCGTGCTGCATCAGGACTGG CTGAACGGCAAAGAATACAAGTGCAAAGTGTCTAATA AGGCCCTGCCTGCTCCAATCGAGAAAACAATTAGCAAG GCAAAAGGGCAGCCCAGGGAACCTCAGGTGTACACTC TGCCTCCAAGCCGCGAGGAAATGACCAAGAACCAGGTC TCCCTGACATGTCTGGTGAAAGGATTCTATCCTAGTG ACATTGCCGTGGAGTGGGAATCAAATGGCCAGCCAGA GAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG ATGGGAGTTTCTTTCTGTATTCCAAGCTGACCGTGGA TAAATCTAGATGGCAGCAGGGAAATGTCTTTAGCTGT TCCGTGATGCATGAGGCACTGCACAACCATTACACCCA GAAATCACTGTCACTGTCCCCAGGAAAAGGCGGGGGA GGCTCTGGAGTGCAGCTGGTCCAGAGCGGAGGAGGAC TGGTGCAGCCAGGAGGGTCACTGAGGCTGAGCTGCGC AGCTTCCGGCTTCACCGTGTCAACAAACTACATGAGCT GGGTCCGCCAGGCACCTGGGAAGGGACTGGAGTGGG TGTCCATCCTGTACGCCGGAGGCGTGACTCGATATGC TGACTCTGTCAAGACTCGGTTCACCATCTCTAGAGATA ACAGTAAGAACACCCTGTTTCTGCAGATGAATGCACTG AGTGCCGAAGACACAGCTATCTACTATTGTGCAAAACA CTACGATTCTGGGTATAGTACAATTGACCATTTTGAT TCTTGGGGCCAGGGGACACTGGTGACTGTCAGCTCCG GCGGGGGAGGCTCTGGGGGAGGCGGGAGTGGAGGCG GGGGATCAGACATCCAGATGACTCAGTCTCCCGATAG TGTGGCCGTCTCCCTGGGGGAGAGGGCTACAATTAAC TGCAAGAGCTCCCAGTCCGTGTTCTACACTTCTAAGAA CAAAAACTATCTGGCATGGTTTCAGCAGAAGCCTGGA CAGCCCCCTAAACTGCTGATCTACTGGGCCTCAACCCG AGAGAGCGGAGTCCCAGACAGATTCTCAGGCAGCGGG TCCGGAACAGATTTTACCCTGACAATTTCTAGTCTGCG GCCTGAAGACGTGGCTGTCTACTATTGTCAGCAGTAC TATAGCACTCCATTCACCTTTGGCCCCGGGACAAAGGT GGATATCAAAGGCGGGGGAGGCTCTGAGGTGCAGCT GGTCGAATCTGGCGGGGACCTGGTGAAGGCAGGAGG CAGTCTGAGGCTGTCATGCGCCGTCTCAGGGCTGAGC TTCAGCTCCTCTGGAATGAACTGGGTGCGCCAGGCAC CAGGCAAAGGACTGGAGTGGATCAGTTCAATTTCTGG CAGTCAGAATTACAAGTACTATGCTGACAGTGTGAAA GGGCGATTCGTGGTCTCCCGGGATAACGCAAGAAATT TTCTGTATCTGCAGATGGACAGCCTGAGAGCCGAAGA TACTGCTGTGTACTTCTGTGTCGGGGGATTTCCCTAT TGGCTGCCCCCTTCCGATTTCTCTGGCTTTCACGTGT GGGGACAGGGCACCACAGTGACCGTCAGCTCCGGCGG GGGAGGCTCTGGGGGAGGCGGGAGTGGAGGCGGGG GATCATCCTACGTGCTGACTCAGCCACCTAGCGTGTCC GTCGCACCTGGACAGACTGCCAGCCTGACCTGCGGAG GAACAAACATCGGGTCTAAGAGTGTGCACTGGTACCA GCAGAAAGCCGGACAGGCTCCCGTCCTGGTGGTCTAT GCCGACAATGATCGGCCATCTGGCGTGCCCGAAAGAT TCTCAGGAAGCAACTCCGGCAATACCGCTACACTGACT ATTTCTAGGGTGGAGGCAGAAGACGAGAGTGATTATT TCTGTCAGGTCTGGGACGGGAACACAGATCATGTGGT CTTTGGAGGCGGGACCAAGCTGACAGTGCTG 153 Ts1GC2a heavy QLQLVQSGTEVKKPGASVKVSCKSSGYVFTSYYLVWVRQ chain aa APGQGLEWMATISPGDVNTSYEQRFQGRVTVTTDASTN TVDMELRSLRSEDTAVYYCARGPRSKPPYLYFALDVWGQ GTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGKGGGGSGVQLVQSGGGL VQPGGSLRLSCAASGFTVSTNYMSWVRQAPGKGLEWVSI LYAGGVTRYADSVKTRFTISRDNSKNTLFLQMNALSAED TAIYYCAKHYDSGYSTIDHFDSWGQGTLVTVSSGGGGSG GGGSGGGGSDIQMTQSPDSVAVSLGERATINCKSSQSVFY TSKNKNYLAWFQQKPGQPPKLLIYWASTRESGVPDRFSG SGSGTDFTLTISSLRPEDVAVYYCQQYYSTPFTFGPGTKV DIKGGGGSEVQLVESGGDLVKAGGSLRLSCAVSGLSFSSS GMNWVRQAPGKGLEWISSISGSQNYKYYADSVKGRFVVS RDNARNFLYLQMDSLRAEDTAVYFCVGGFPYWLPPSDF SGFHVWGQGTTVTVSSGGGGSGGGGSGGGGSSYVLTQP PSVSVAPGQTASLTCGGTNIGSKSVHWYQQKAGQAPVLV VYADNDRPSGVPERFSGSNSGNTATLTISRVEAEDESDYF CQVWDGNTDHVVFGGGTKLTVL 154 Ts1GC2a heavy CAGCTGCAGCTGGTCCAGTCAGGCACAGAGGTCAAAA AGCCAGGAGCATCAGTGAAGGTGTCTTGTAAGTCATC AGGATACGTGTTCACCTCTTACTATCTGGTGTGGGTC CGGCAGGCACCAGGACAGGGACTGGAGTGGATGGCCA CAATCTCTCCCGGAGACGTGAACACTAGTTACGAACAG CGATTCCAGGGCAGAGTGACCGTCACCACAGACGCTTC AACTAATACCGTGGATATGGAGCTGCGGAGCCTGAGA TCCGAAGATACAGCCGTCTACTATTGCGCTAGGGGGC CCCGCAGCAAGCCTCCTTATCTGTATTTTGCTCTGGAT GTGTGGGGGCAGGGGACCGCTGTCACCGTGTCAAGCG CGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCC TCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGG GCTGCCTGGTCAAGGACTACTTCCCCGAACCTGTGACG GTCTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT GGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC CCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAA TCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGC ACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGA GGATCCTGAAGTCAAGTTCAACTGGTACGTGGATGGC GTCGAGGTGCATAATGCCAAGACAAAACCCCGGGAGG AACAGTACAACTCAACTTATAGAGTCGTGAGCGTCCTG ACCGTGCTGCATCAGGACTGGCTGAACGGCAAAGAAT ACAAGTGCAAAGTGTCTAATAAGGCCCTGCCTGCTCCA ATCGAGAAAACAATTAGCAAGGCAAAAGGGCAGCCCAG GGAACCTCAGGTGTACACTCTGCCTCCAAGCCGCGAGG AAATGACCAAGAACCAGGTCTCCCTGACATGTCTGGTG AAAGGATTCTATCCTAGTGACATTGCCGTGGAGTGGG AATCAAATGGCCAGCCAGAGAACAATTACAAGACCACA CCCCCTGTGCTGGACTCTGATGGGAGTTTCTTTCTGT ATTCCAAGCTGACCGTGGATAAATCTAGATGGCAGCA GGGAAATGTCTTTAGCTGTTCCGTGATGCATGAGGCA CTGCACAACCATTACACCCAGAAATCACTGTCACTGTC CCCAGGAAAAGGCGGGGGAGGCTCTGGAGTGCAGCTG GTCCAGAGCGGAGGAGGACTGGTGCAGCCAGGAGGG TCACTGAGGCTGAGCTGCGCAGCTTCCGGCTTCACCG TGTCAACAAACTACATGAGCTGGGTCCGCCAGGCACCT GGGAAGGGACTGGAGTGGGTGTCCATCCTGTACGCCG GAGGCGTGACTCGATATGCTGACTCTGTCAAGACTCG GTTCACCATCTCTAGAGATAACAGTAAGAACACCCTGT TTCTGCAGATGAATGCACTGAGTGCCGAAGACACAGC TATCTACTATTGTGCAAAACACTACGATTCTGGGTAT AGTACAATTGACCATTTTGATTCTTGGGGCCAGGGGA CACTGGTGACTGTCAGCTCCGGCGGGGGAGGCTCTGG GGGAGGCGGGAGTGGAGGCGGGGGATCAGACATCCA GATGACTCAGTCTCCCGATAGTGTGGCCGTCTCCCTG GGGGAGAGGGCTACAATTAACTGCAAGAGCTCCCAGT CCGTGTTCTACACTTCTAAGAACAAAAACTATCTGGCA TGGTTTCAGCAGAAGCCTGGACAGCCCCCTAAACTGCT GATCTACTGGGCCTCAACCCGAGAGAGCGGAGTCCCA GACAGATTCTCAGGCAGCGGGTCCGGAACAGATTTTA CCCTGACAATTTCTAGTCTGCGGCCTGAAGACGTGGC TGTCTACTATTGTCAGCAGTACTATAGCACTCCATTCA CCTTTGGCCCCGGGACAAAGGTGGATATCAAAGGCGG GGGAGGCTCTGAGGTGCAGCTGGTCGAATCTGGCGG GGACCTGGTGAAGGCAGGAGGCAGTCTGAGGCTGTCA TGCGCCGTCTCAGGGCTGAGCTTCAGCTCCTCTGGAA TGAACTGGGTGCGCCAGGCACCAGGCAAAGGACTGGA GTGGATCAGTTCAATTTCTGGCAGTCAGAATTACAAG TACTATGCTGACAGTGTGAAAGGGCGATTCGTGGTCT CCCGGGATAACGCAAGAAATTTTCTGTATCTGCAGAT GGACAGCCTGAGAGCCGAAGATACTGCTGTGTACTTC TGTGTCGGGGGATTTCCCTATTGGCTGCCCCCTTCCG ATTTCTCTGGCTTTCACGTGTGGGGACAGGGCACCAC AGTGACCGTCAGCTCCGGCGGGGGAGGCTCTGGGGG AGGCGGGAGTGGAGGCGGGGGATCATCCTACGTGCT GACTCAGCCACCTAGCGTGTCCGTCGCACCTGGACAGA CTGCCAGCCTGACCTGCGGAGGAACAAACATCGGGTC TAAGAGTGTGCACTGGTACCAGCAGAAAGCCGGACAG GCTCCCGTCCTGGTGGTCTATGCCGACAATGATCGGC CATCTGGCGTGCCCGAAAGATTCTCAGGAAGCAACTCC GGCAATACCGCTACACTGACTATTTCTAGGGTGGAGG CAGAAGACGAGAGTGATTATTTCTGTCAGGTCTGGGA CGGGAACACAGATCATGTGGTCTTTGGAGGCGGGACC AAGCTGACAGTGCTG 155 Ts2GC2b/Bs1GC2a EVQLVESGGDLVKAGGSLRLSCAVSGLSFSSSGMNWVRQ heavy chain aa APGKGLEWISSISGSQNYKYYADSVKGRFVVSRDNARNFL YLQMDSLRAEDTAVYFCVGGFPYWLPPSDFSGFHVWGQ GTTVTVSSGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGQ TASLTCGGTNIGSKSVHWYQQKAGQAPVLVVYADNDRPS GVPERFSGSNSGNTATLTISRVEAEDESDYFCQVWDGNT DHVVFGGGTKLTVLGGGGSQLQLVQSGTEVKKPGASVK VSCKSSGYVFTSYYLVWVRQAPGQGLEWMATISPGDVN TSYEQRFQGRVTVTTDASTNTVDMELRSLRSEDTAVYYC ARGPRSKPPYLYFALDVWGQGTAVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK 156 Ts2GC2b/Bs1GC2a GAGGTGCAGCTGGTGGAAAGCGGAGGGGATCTGGTG heavy chain nucl AAAGCAGGAGGGAGCCTGAGACTGTCATGCGCCGTGA GCGGGCTGTCATTCAGCTCCTCTGGCATGAACTGGG TGCGACAGGCTCCTGGAAAGGGACTGGAGTGGATCAG TTCAATTAGCGGGTCCCAGAATTACAAGTACTATGCA GACTCTGTCAAAGGAAGGTTCGTGGTCAGCCGGGATA ACGCCAGAAATTTTCTGTATCTGCAGATGGACAGCCT GCGCGCCGAAGATACCGCCGTGTACTTCTGCGTCGGC GGGTTTCCCTATTGGCTGCCTCCAAGCGATTTCAGC GGATTTCATGTCTGGGGGCAGGGAACTACAGTGACC GTCTCATCAGGCGGGGGAGGCTCTGGGGGAGGCGGG AGTGGAGGCGGGGGATCATCTTACGTCCTGACCCAGC CACCTAGCGTGAGCGTCGCACCAGGGCAGACAGCTTCA CTGACTTGCGGAGGCACAAACATTGGCAGCAAGAGCG TGCACTGGTACCAGCAGAAAGCCGGACAGGCTCCCGTC CTGGTGGTCTATGCTGACAACGATCGGCCCTCTGGCG TGCCTGAAAGATTCAGCGGCTCCAACTCTGGGAATACC GCAACACTGACCATCAGTAGGGTCGAGGCCGAAGACG AGTCAGATTACTTTTGCCAGGTGTGGGACGGCAATA CTGACCATGTCGTGTTCGGCGGCGGGACCAAACTGAC TGTGCTGGGCGGGGGAGGCTCTCAGCTGCAGCTGGT CCAGTCAGGCACAGAAGTCAAAAAACCCGGCGCAAGCG TGAAGGTCTCATGTAAATCATCAGGATACGTCTTTAC CTCTTACTATCTGGTGTGGGTCCGGCAGGCACCAGGA CAGGGACTGGAGTGGATGGCCACAATCTCTCCCGGAG ACGTGAACACTAGTTACGAACAGCGATTCCAGGGCAG AGTGACCGTCACCACAGACGCTTCAACTAATACCGTGG ATATGGAGCTGCGGAGCCTGAGATCCGAAGATACAGC CGTCTACTATTGCGCTAGGGGGCCCCGCAGCAAGCCT CCTTATCTGTACTTCGCTCTGGATGTCTGGGGGCAG GGGACCGCCGTCACCGTCTCAAGCGCGTCGACCAAGG GCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA AGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAAC TCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGC TGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCG TGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAA GGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCT GGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA TGCGTGGTGGTGGACGTGAGCCACGAGGATCCTGAG GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGC ATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAA CCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG GTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAA GAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCT ATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGC TGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCT TCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAA 157 Ts2GC2b/Bs2GC1c GVQLVQSGGGLVQPGGSLRLSCAASGFTVSTNYMSWVR light chain aa QAPGKGLEWVSILYAGGVTRYADSVKTRFTISRDNSKNT LFLQMNALSAEDTAIYYCAKHYDSGYSTIDHFDSWGQGT LVTVSSGGGGSGGGGSGGGGSDIQMTQSPDSVAVSLGER ATINCKSSQSVFYTSKNKNYLAWFQQKPGQPPKLLIYWAS TRESGVPDRFSGSGSGTDFTLTISSLRPEDVAVYYCQQYY STPFTFGPGTKVDIKGGGGSEIVLTQSPGTLSLSPGETAIL SCRASQSVSSSLLAWYQQKPGQAPRLLIYGASNRATGIRG RFSGSGSGTDFTLTISRLEPEDFVLYYCQHYGSRVTFGQG TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 158 Ts2GC2b/Bs2GC1c GGCGTCCAGCTGGTGCAGAGCGGAGGGGGCCTGGTG light chain nucl CAGCCTGGCGGGTCCCTGAGACTGAGTTGTGCCGCAA GTGGCTTTACTGTCTCTACAAACTACATGTCTTGGG TGAGGCAGGCACCTGGAAAGGGACTGGAGTGGGTCTC AATCCTGTACGCTGGCGGGGTGACCCGGTATGCAGA CAGCGTCAAGACCCGGTTCACAATTAGCAGAGATAACT CCAAAAATACTCTGTTTCTGCAGATGAATGCCCTGTCC GCTGAAGACACCGCAATCTACTATTGCGCCAAACACTA TGATAGTGGGTATAGCACAATCGACCATTTTGACAG CTGGGGACAGGGAACTCTGGTGACAGTCTCATCAGGC GGGGGAGGCTCTGGGGGAGGCGGGAGTGGAGGCGG GGGATCAGACATTCAGATGACCCAGAGTCCTGATTCC GTGGCTGTCTCACTGGGGGAGCGAGCAACTATTAACT GCAAGTCTTCACAGAGCGTGTTCTACACCAGTAAGAA CAAAAACTATCTGGCCTGGTTTCAGCAGAAGCCAGGC CAGCCCCCTAAACTGCTGATCTACTGGGCTAGCACTA GAGAGTCTGGAGTGCCAGACAGATTCTCTGGCAGTGG GTCAGGAACCGACTTCACCCTGACAATTAGCTCCCTGA GGCCCGAAGACGTGGCCGTCTACTATTGTCAGCAGTA TTACAGCACCCCATTCACATTCGGCCCTGGAACCAAAG TGGATATTAAGGGCGGGGGAGGCTCTGAAATCGTGC TGACCCAGTCTCCTGGAACTCTGTCTCTGTCACCTGGC GAAACCGCAATCCTGTCCTGTAGGGCAAGTCAGTCTG TGAGCTCCTCTCTGCTGGCATGGTACCAGCAGAAGCC CGGACAGGCCCCTAGGCTGCTGATCTATGGCGCCTCC AACCGCGCTACTGGCATTCGGGGGAGATTCAGTGGCT CAGGGAGCGGAACCGACTTTACCCTGACAATCAGCCGG CTGGAGCCCGAAGATTTCGTGCTGTATTACTGTCAGC ACTATGGCAGCAGGGTCACTTTTGGGCAGGGGACTA AACTGGAGATTAAAcgTacGgtggctgcaccatctgtcttcatcttcccgcc atctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatc ccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccag gagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgac gctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagg gcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 159 Ts2GC2c/Bs1GC3a EVQLVESGGDLVKAGGSLRLSCAVSGLSFSSSGMNWVRQ heavy chain aa APGKGLEWISSISGSQNYKYYADSVKGRFVVSRDNARNFL YLQMDSLRAEDTAVYFCVGGFPYWLPPSDFSGFHVWGQ GTTVTVSSGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGQ TASLTCGGTNIGSKSVHWYQQKAGQAPVLVVYADNDRPS GVPERFSGSNSGNTATLTISRVEAEDESDYFCQVWDGNT DHVVFGGGTKLTVLGGGGSGVQLVQSGGGLVQPGGSLR LSCAASGFTVSTNYMSWVRQAPGKGLEWVSILYAGGVT RYADSVKTRFTISRDNSKNTLFLQMNALSAEDTAIYYCAK HYDSGYSTIDHFDSWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 160 Ts2GC2c/Bs1GC3a GAGGTGCAGCTGGTGGAAAGTGGGGGCGATCTGGTC heavy chain nucl AAAGCCGGAGGGTCTCTGCGACTGTCTTGTGCTGTGA GCGGCCTGTCCTTCAGCTCCTCTGGCATGAACTGGG TGCGACAGGCTCCTGGAAAGGGACTGGAGTGGATCAG TTCAATTAGCGGGTCCCAGAATTACAAGTACTATGCA GACTCTGTCAAAGGAAGGTTCGTGGTCAGCCGGGATA ACGCCAGAAATTTTCTGTATCTGCAGATGGACAGCCT GCGCGCCGAAGATACCGCCGTGTACTTCTGCGTCGGC GGGTTTCCCTATTGGCTGCCTCCAAGCGACTTTTCA GGGTTTCATGTCTGGGGGCAGGGAACTACCGTGACC GTCTCATCTGGCGGGGGAGGCTCTGGGGGAGGCGGG AGTGGAGGCGGGGGATCATCCTACGTCCTGACTCAGC CACCTAGCGTGTCCGTCGCACCTGGGCAGACAGCATCA CTGACTTGCGGGGGAACCAACATCGGCAGCAAGAGCG TGCACTGGTACCAGCAGAAAGCCGGACAGGCTCCCGTC CTGGTGGTCTATGCTGACAACGATCGGCCCTCTGGCG TGCCTGAAAGATTCAGCGGCTCCAACTCTGGGAATACC GCAACACTGACCATCAGTAGGGTCGAGGCCGAAGACG AGTCAGATTACTTTTGCCAGGTCTGGGATGGGAATA CTGACCACGTCGTCTTCGGAGGCGGAACCAAACTGAC TGTCCTGGGCGGGGGAGGCTCTGGCGTGCAGCTGGT GCAGAGCGGCGGCGGCCTGGTGCAGCCTGGAGGGTCA CTGAGACTGTCATGCGCAGCAAGCGGGTTTACTGTG TCTACAAACTACATGTCTTGGGTGAGGCAGGCACCTG GAAAGGGACTGGAGTGGGTCTCAATCCTGTACGCTG GCGGGGTGACCCGGTATGCAGACAGCGTCAAGACCCG GTTCACAATTAGCAGAGATAACTCCAAAAATACTCTGT TTCTGCAGATGAATGCCCTGTCCGCTGAAGACACCGCA ATCTACTATTGCGCCAAACACTATGATAGTGGGTACT CCACTATTGACCATTTTGACTCTTGGGGGCAGGGGA CTCTGGTGACTGTCTCTTCAGCGTCGACCAAGGGCCCA TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTC TGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTC CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACA TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTG GACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCA CACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGAC ACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT GGTGGTGGACGTGAGCCACGAGGATCCTGAGGTCAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGA CTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC CCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGC GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC GACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGA CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCA GAAGAGCCTCTCCCTGTCCCCGGGTAAA 161 Ts2GC2c/Bs2GC1d QLQLVQSGTEVKKPGASVKVSCKSSGYVFTSYYLVWVRQ light chain aa APGQGLEWMATISPGDVNTSYEQRFQGRVTVTTDASTN TVDMELRSLRSEDTAVYYCARGPRSKPPYLYFALDVWGQ GTAVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG ETAILSCRASQSVSSSLLAWYQQKPGQAPRLLIYGASNRAT GIRGRFSGSGSGTDFTLTISRLEPEDFVLYYCQHYGSRVT FGQGTKLEIKGGGGSDIQMTQSPDSVAVSLGERATINCKS SQSVFYTSKNKNYLAWFQQKPGQPPKLLIYWASTRESGV PDRFSGSGSGTDFTLTISSLRPEDVAVYYCQQYYSTPFTF GPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 162 Ts2GC2c/Bs2GC1d CAGCTGCAGCTGGTCCAGAGCGGCACAGAGGTGAAAA light chain nucl AGCCAGGAGCATCAGTCAAAGTGTCTTGTAAGTCATC AGGATACGTCTTCACCTCTTACTATCTGGTGTGGGT CCGGCAGGCACCAGGACAGGGACTGGAGTGGATGGCC ACAATCTCTCCCGGAGACGTGAACACTAGTTACGAAC AGCGATTCCAGGGCAGAGTGACCGTCACCACAGACGCT TCAACTAATACCGTGGATATGGAGCTGCGGAGCCTGA GATCCGAAGATACAGCCGTCTACTATTGCGCTAGGGG GCCCCGCAGCAAGCCTCCTTATCTGTATTTCGCTCT GGATGTCTGGGGGCAGGGAACAGCAGTCACCGTCTCT TCTGGCGGGGGAGGCTCTGGGGGAGGCGGGAGTGGA GGCGGGGGATCAGAAATCGTCCTGACCCAGTCACCTG GCACCCTGAGTCTGAGTCCTGGCGAAACAGCAATCCTG TCTTGTCGGGCTTCACAGTCCGTGAGCTCCTCTCTGC TGGCATGGTACCAGCAGAAGCCCGGACAGGCCCCTAG GCTGCTGATCTATGGCGCCTCCAACCGCGCTACTGGC ATTCGGGGGAGATTCAGTGGCTCAGGGAGCGGAACCG ACTTTACCCTGACAATCAGCCGGCTGGAGCCCGAAGAT TTCGTGCTGTACTACTGTCAGCATTATGGGTCACGG GTCACTTTTGGGCAGGGGACTAAACTGGAAATCAAGG GCGGGGGAGGCTCTGACATTCAGATGACCCAGAGTCC TGACAGCGTGGCCGTCTCACTGGGGGAAAGGGCTACT ATCAATTGTAAAAGTTCACAGTCCGTCTTCTACACCA GTAAGAACAAAAACTATCTGGCCTGGTTTCAGCAGAA GCCAGGCCAGCCCCCTAAACTGCTGATCTACTGGGCT AGCACTAGAGAGTCTGGAGTGCCAGACAGATTCTCTG GCAGTGGGTCAGGAACCGACTTCACCCTGACAATTAGC TCCCTGAGGCCCGAAGACGTGGCCGTCTATTATTGTC AGCAGTATTATTCTACCCCCTTCACATTCGGACCTGG GACTAAAGTGGATATCAAACGTACGGTGGCTGCACCA TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGG ATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAG CAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GT 163 Ts3GC2d heavy EVQLVESGGDLVKAGGSLRLSCAVSGLSFSSSGMNWVRQ chain aa APGKGLEWISSISGSQNYKYYADSVKGRFVVSRDNARNFL YLQMDSLRAEDTAVYFCVGGFPYWLPPSDFSGFHVWGQ GTTVTVSSGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGQ TASLTCGGTNIGSKSVHWYQQKAGQAPVLVVYADNDRPS GVPERFSGSNSGNTATLTISRVEAEDESDYFCQVWDGNT DHVVFGGGTKLTVLGGGGSQLQLVQSGTEVKKPGASVK VSCKSSGYVFTSYYLVWVRQAPGQGLEWMATISPGDVN TSYEQRFQGRVTVTTDASTNTVDMELRSLRSEDTAVYYC ARGPRSKPPYLYFALDVWGQGTAVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKGGGGSGVQLVQSGGGLVQPGGSLRLSCAASGFTV STNYMSWVRQAPGKGLEWVSILYAGGVTRYADSVKTRF TISRDNSKNTLFLQMNALSAEDTAIYYCAKHYDSGYSTID HFDSWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSP DSVAVSLGERATINCKSSQSVFYTSKNKNYLAWFQQKPG QPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLRPED VAVYYCQQYYSTPFTFGPGTKVDIK 164 Ts3GC2d heavy GAGGTGCAGCTGGTGGAAAGCGGAGGGGATCTGGTG chain nucl AAAGCAGGAGGGAGCCTGAGACTGTCATGCGCCGTGA GCGGGCTGTCATTCAGCTCCTCTGGCATGAACTGGG TGCGACAGGCTCCTGGAAAGGGACTGGAGTGGATCAG TTCAATTAGCGGGTCCCAGAATTACAAGTACTATGCA GACTCTGTCAAAGGAAGGTTCGTGGTCAGCCGGGATA ACGCCAGAAATTTTCTGTATCTGCAGATGGACAGCCT GCGCGCCGAAGATACCGCCGTGTACTTCTGCGTCGGC GGGTTTCCCTATTGGCTGCCTCCAAGCGATTTCAGC GGATTTCATGTCTGGGGGCAGGGAACTACAGTGACC GTCTCATCAGGCGGGGGAGGCTCTGGGGGAGGCGGG AGTGGAGGCGGGGGATCATCTTACGTCCTGACCCAGC CACCTAGCGTGAGCGTCGCACCAGGGCAGACAGCTTCA CTGACTTGCGGAGGCACAAACATTGGCAGCAAGAGCG TGCACTGGTACCAGCAGAAAGCCGGACAGGCTCCCGTC CTGGTGGTCTATGCTGACAACGATCGGCCCTCTGGCG TGCCTGAAAGATTCAGCGGCTCCAACTCTGGGAATACC GCAACACTGACCATCAGTAGGGTCGAGGCCGAAGACG AGTCAGATTACTTTTGCCAGGTGTGGGACGGCAATA CTGACCATGTCGTGTTCGGCGGCGGGACCAAACTGAC TGTGCTGGGCGGGGGAGGCTCTCAGCTGCAGCTGGT CCAGTCAGGCACAGAAGTCAAAAAACCCGGCGCAAGCG TGAAGGTCTCATGTAAATCATCAGGATACGTCTTTAC CTCTTACTATCTGGTGTGGGTCCGGCAGGCACCAGGA CAGGGACTGGAGTGGATGGCCACAATCTCTCCCGGAG ACGTGAACACTAGTTACGAACAGCGATTCCAGGGCAG AGTGACCGTCACCACAGACGCTTCAACTAATACCGTGG ATATGGAGCTGCGGAGCCTGAGATCCGAAGATACAGC CGTCTACTATTGCGCTAGGGGGCCCCGCAGCAAGCCT CCTTATCTGTACTTCGCTCTGGATGTCTGGGGGCAG GGGACCGCCGTCACCGTCTCAAGCGCGTCGACCAAGG GCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCA AGGACTACTTCCCCGAACCTGTGACGGTCTCGTGGAAC TCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGC TGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCG TGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAA GGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAA ACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCT GGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCA AGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA TGCGTGGTGGTGGACGTGAGCCACGAGGATCCTGAAG TCAAGTTCAACTGGTACGTGGATGGCGTCGAGGTGCA TAATGCCAAGACAAAACCCCGGGAGGAACAGTACAACT CAACTTATAGAGTCGTGAGCGTCCTGACCGTGCTGCA TCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAA GTGTCTAATAAGGCCCTGCCTGCTCCAATCGAGAAAAC AATTAGCAAGGCAAAAGGGCAGCCCAGGGAACCTCAG GTGTACACTCTGCCTCCAAGCCGCGAGGAAATGACCAA GAACCAGGTCTCCCTGACATGTCTGGTGAAAGGATTC TATCCTAGTGACATTGCCGTGGAGTGGGAATCAAATG GCCAGCCAGAGAACAATTACAAGACCACACCCCCTGTG CTGGACTCTGATGGGAGTTTCTTTCTGTATTCCAAGC TGACCGTGGATAAATCTAGATGGCAGCAGGGAAATGT CTTTAGCTGTTCCGTGATGCATGAGGCACTGCACAAC CATTACACCCAGAAATCACTGTCACTGTCCCCAGGAAA AGGCGGGGGAGGCTCTGGCGTGCAGCTGGTCCAGAG CGGAGGCGGACTGGTCCAGCCCGGCGGATCACTGAGA CTGTCATGTGCCGCAAGCGGGTTTACCGTCTCTACAA ACTACATGTCTTGGGTGAGGCAGGCACCTGGAAAGGG ACTGGAGTGGGTCTCAATCCTGTACGCTGGCGGGGT GACCCGGTATGCAGACAGCGTCAAGACCCGGTTCACAA TTAGCAGAGATAACTCCAAAAATACTCTGTTTCTGCAG ATGAATGCCCTGTCCGCTGAAGACACCGCAATCTACTA TTGCGCCAAACACTATGATAGTGGGTACAGTACCAT TGACCATTTCGATAGCTGGGGGCAGGGGACTCTGGT GACCGTCTCATCAGGCGGGGGAGGCTCTGGGGGAGG CGGGAGTGGAGGCGGGGGATCAGATATTCAGATGACC CAGAGTCCTGATTCCGTCGCTGTCTCACTGGGAGAAA GGGCAACCATTAACTGTAAAAGCTCACAGAGTGTCTT CTACACCAGTAAGAACAAAAACTATCTGGCCTGGTTT CAGCAGAAGCCAGGCCAGCCCCCTAAACTGCTGATCTA CTGGGCTAGCACTAGAGAGTCTGGAGTGCCAGACAGA TTCTCTGGCAGTGGGTCAGGAACCGACTTCACCCTGA CAATTAGCTCCCTGAGGCCCGAAGACGTGGCCGTCTAC TATTGTCAGCAGTATTATTCAACACCCTTCACATTCG GACCAGGAACAAAAGTGGATATTAAG 165 Ts3GC2e heavy EVQLVESGGDLVKAGGSLRLSCAVSGLSFSSSGMNWVRQ chain aa APGKGLEWISSISGSQNYKYYADSVKGRFVVSRDNARNFL YLQMDSLRAEDTAVYFCVGGFPYWLPPSDFSGFHVWGQ GTTVTVSSGGGGSGGGGSGGGGSSYVLTQPPSVSVAPGQ TASLTCGGTNIGSKSVHWYQQKAGQAPVLVVYADNDRPS GVPERFSGSNSGNTATLTISRVEAEDESDYFCQVWDGNT DHVVFGGGTKLTVLGGGGSGVQLVQSGGGLVQPGGSLR LSCAASGFTVSTNYMSWVRQAPGKGLEWVSILYAGGVT RYADSVKTRFTISRDNSKNTLFLQMNALSAEDTAIYYCAK HYDSGYSTIDHFDSWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKG GGGSQLQLVQSGTEVKKPGASVKVSCKSSGYVFTSYYLV WVRQAPGQGLEWMATISPGDVNTSYEQRFQGRVTVTT DASTNTVDMELRSLRSEDTAVYYCARGPRSKPPYLYFAL DVWGQGTAVTVSSGGGGSGGGGSGGGGSEIVLTQSPGT LSLSPGETAILSCRASQSVSSSLLAWYQQKPGQAPRLLIYG ASNRATGIRGRFSGSGSGTDFTLTISRLEPEDFVLYYCQH YGSRVTFGQGTKLEIK 166 Ts3GC2e heavy GAGGTGCAGCTGGTGGAAAGTGGGGGCGATCTGGTC chain nucl AAAGCCGGAGGGTCTCTGCGACTGTCTTGTGCTGTGA GCGGCCTGTCCTTCAGCTCCTCTGGCATGAACTGGG TGCGACAGGCTCCTGGAAAGGGACTGGAGTGGATCAG TTCAATTAGCGGGTCCCAGAATTACAAGTACTATGCA GACTCTGTCAAAGGAAGGTTCGTGGTCAGCCGGGATA ACGCCAGAAATTTTCTGTATCTGCAGATGGACAGCCT GCGCGCCGAAGATACCGCCGTGTACTTCTGCGTCGGC GGGTTTCCCTATTGGCTGCCTCCAAGCGACTTTTCA GGGTTTCATGTCTGGGGGCAGGGAACTACCGTGACC GTCTCATCTGGCGGGGGAGGCTCTGGGGGAGGCGGG AGTGGAGGCGGGGGATCATCCTACGTCCTGACTCAGC CACCTAGCGTGTCCGTCGCACCTGGGCAGACAGCATCA CTGACTTGCGGGGGAACCAACATCGGCAGCAAGAGCG TGCACTGGTACCAGCAGAAAGCCGGACAGGCTCCCGTC CTGGTGGTCTATGCTGACAACGATCGGCCCTCTGGCG TGCCTGAAAGATTCAGCGGCTCCAACTCTGGGAATACC GCAACACTGACCATCAGTAGGGTCGAGGCCGAAGACG AGTCAGATTACTTTTGCCAGGTCTGGGATGGGAATA CTGACCACGTCGTCTTCGGAGGCGGAACCAAACTGAC TGTCCTGGGCGGGGGAGGCTCTGGCGTGCAGCTGGT GCAGAGCGGCGGCGGCCTGGTGCAGCCTGGAGGGTCA CTGAGACTGTCATGCGCAGCAAGCGGGTTTACTGTG TCTACAAACTACATGTCTTGGGTGAGGCAGGCACCTG GAAAGGGACTGGAGTGGGTCTCAATCCTGTACGCTG GCGGGGTGACCCGGTATGCAGACAGCGTCAAGACCCG GTTCACAATTAGCAGAGATAACTCCAAAAATACTCTGT TTCTGCAGATGAATGCCCTGTCCGCTGAAGACACCGCA ATCTACTATTGCGCCAAACACTATGATAGTGGGTACT CCACTATTGACCATTTTGACTCTTGGGGGCAGGGGA CTCTGGTGACTGTCTCTTCAGCGTCGACCAAGGGCCCA TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTC TGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC TACTTCCCCGAACCTGTGACGGTCTCGTGGAACTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTC CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACA TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTG GACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCA CACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGAC ACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT GGTGGTGGACGTGAGCCACGAGGATCCTGAAGTCAAG TTCAACTGGTACGTGGATGGCGTCGAGGTGCATAATG CCAAGACAAAACCCCGGGAGGAACAGTACAACTCAACT TATAGAGTCGTGAGCGTCCTGACCGTGCTGCATCAGG ACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTGTC TAATAAGGCCCTGCCTGCTCCAATCGAGAAAACAATTA GCAAGGCAAAAGGGCAGCCCAGGGAACCTCAGGTGTA CACTCTGCCTCCAAGCCGCGAGGAAATGACCAAGAACC AGGTCTCCCTGACATGTCTGGTGAAAGGATTCTATCC TAGTGACATTGCCGTGGAGTGGGAATCAAATGGCCAG CCAGAGAACAATTACAAGACCACACCCCCTGTGCTGGA CTCTGATGGGAGTTTCTTTCTGTATTCCAAGCTGACC GTGGATAAATCTAGATGGCAGCAGGGAAATGTCTTTA GCTGTTCCGTGATGCATGAGGCACTGCACAACCATTAC ACCCAGAAATCACTGTCACTGTCCCCAGGAAAAGGCGG GGGAGGCTCTCAGCTGCAGCTGGTCCAGAGCGGAACC GAAGTGAAGAAACCCGGCGCAAGCGTCAAAGTCTCAT GCAAATCAAGCGGATACGTCTTCACCTCTTACTATCT GGTGTGGGTCCGGCAGGCACCAGGACAGGGACTGGA GTGGATGGCCACAATCTCTCCCGGAGACGTGAACACT AGTTACGAACAGCGATTCCAGGGCAGAGTGACCGTCA CCACAGACGCTTCAACTAATACCGTGGATATGGAGCTG CGGAGCCTGAGATCCGAAGATACAGCCGTCTACTATT GCGCTAGGGGGCCCCGCAGCAAGCCTCCTTATCTGT ATTTCGCTCTGGATGTCTGGGGGCAGGGAACAGCAG TCACCGTCTCAAGCGGCGGGGGAGGCTCTGGGGGAG GCGGGAGTGGAGGCGGGGGATCAGAGATTGTCCTGA CCCAGTCACCTGGCACCCTGAGCCTGAGTCCTGGAGAG ACCGCTATTCTGTCTTGTCGGGCATCACAGTCCGTGA GCTCCTCTCTGCTGGCATGGTACCAGCAGAAGCCCGG ACAGGCCCCTAGGCTGCTGATCTATGGCGCCTCCAAC CGCGCTACTGGCATTCGGGGGAGATTCAGTGGCTCAG GGAGCGGAACCGACTTTACCCTGACAATCAGCCGGCT GGAGCCCGAAGATTTCGTGCTGTACTATTGTCAGCAT TATGGAAGCAGGGTCACCTTCGGACAGGGAACTAAAC TGGAAATCAAG 167 Bs3GC1a heavy QLQLVQSGTEVKKPGASVKVSCKSSGYVFTSYYLVWVRQ chain aa APGQGLEWMATISPGDVNTSYEQRFQGRVTVTTDASTN TVDMELRSLRSEDTAVYYCARGPRSKPPYLYFALDVWGQ GTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGKGGGGSGVQLVQSGGGL VQPGGSLRLSCAASGFTVSTNYMSWVRQAPGKGLEWVSI LYAGGVTRYADSVKTRFTISRDNSKNTLFLQMNALSAED TAIYYCAKHYDSGYSTIDHFDSWGQGTLVTVSSGGGGSG GGGSGGGGSDIQMTQSPDSVAVSLGERATINCKSSQSVFY TSKNKNYLAWFQQKPGQPPKLLIYWASTRESGVPDRFSG SGSGTDFTLTISSLRPEDVAVYYCQQYYSTPFTFGPGTKV DIK 168 Bs3GC1a heavy CAGCTGCAGCTGGTCCAGTCAGGCACAGAGGTCAAAA chain nucl AGCCAGGAGCATCAGTGAAGGTGTCTTGTAAGTCATC AGGATACGTGTTCACCTCTTACTATCTGGTGTGGGT CCGGCAGGCACCAGGACAGGGACTGGAGTGGATGGCC ACAATCTCTCCCGGAGACGTGAACACTAGTTACGAAC AGCGATTCCAGGGCAGAGTGACCGTCACCACAGACGCT TCAACTAATACCGTGGATATGGAGCTGCGGAGCCTGA GATCCGAAGATACAGCCGTCTACTATTGCGCTAGGGG GCCCCGCAGCAAGCCTCCTTATCTGTATTTTGCTCT GGATGTGTGGGGGCAGGGGACCGCTGTCACCGTGTC AAGCGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGG CACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGT GACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACT CTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA GCTTGGGCACCCAGACCTACATCTGCAACGTGAATCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCC CAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCC ACGAGGATCCTGAAGTCAAGTTCAACTGGTACGTGGA TGGCGTCGAGGTGCATAATGCCAAGACAAAACCCCGG GAGGAACAGTACAACTCAACTTATAGAGTCGTGAGCG TCCTGACCGTGCTGCATCAGGACTGGCTGAACGGCAA AGAATACAAGTGCAAAGTGTCTAATAAGGCCCTGCCT GCTCCAATCGAGAAAACAATTAGCAAGGCAAAAGGGCA GCCCAGGGAACCTCAGGTGTACACTCTGCCTCCAAGCC GCGAGGAAATGACCAAGAACCAGGTCTCCCTGACATG TCTGGTGAAAGGATTCTATCCTAGTGACATTGCCGTG GAGTGGGAATCAAATGGCCAGCCAGAGAACAATTACA AGACCACACCCCCTGTGCTGGACTCTGATGGGAGTTT CTTTCTGTATTCCAAGCTGACCGTGGATAAATCTAGA TGGCAGCAGGGAAATGTCTTTAGCTGTTCCGTGATGC ATGAGGCACTGCACAACCATTACACCCAGAAATCACTG TCACTGTCCCCAGGAAAAGGCGGGGGAGGCTCTGGCG TGCAGCTGGTCCAGAGCGGAGGCGGACTGGTCCAGCC CGGCGGATCACTGAGACTGTCATGTGCCGCAAGCGGG TTTACCGTCTCTACAAACTACATGTCTTGGGTGAGGC AGGCACCTGGAAAGGGACTGGAGTGGGTCTCAATCCT GTACGCTGGCGGGGTGACCCGGTATGCAGACAGCGT CAAGACCCGGTTCACAATTAGCAGAGATAACTCCAAAA ATACTCTGTTTCTGCAGATGAATGCCCTGTCCGCTGA AGACACCGCAATCTACTATTGCGCCAAACACTATGATA GTGGGTACAGTACCATTGACCATTTCGATAGCTGGG GGCAGGGGACTCTGGTGACCGTCTCATCAGGCGGGGG AGGCTCTGGGGGAGGCGGGAGTGGAGGCGGGGGATC AGATATTCAGATGACCCAGAGTCCTGATTCCGTCGCT GTCTCACTGGGAGAAAGGGCAACCATTAACTGTAAAA GCTCACAGAGTGTCTTCTACACCAGTAAGAACAAAAA CTATCTGGCCTGGTTTCAGCAGAAGCCAGGCCAGCCCC CTAAACTGCTGATCTACTGGGCTAGCACTAGAGAGTC TGGAGTGCCAGACAGATTCTCTGGCAGTGGGTCAGGA ACCGACTTCACCCTGACAATTAGCTCCCTGAGGCCCGA AGACGTGGCCGTCTACTATTGTCAGCAGTATTATTCA ACACCCTTCACATTCGGACCAGGAACAAAAGTGGATA TTAAG 169 Bs3GC1b heavy GVQLVQSGGGLVQPGGSLRLSCAASGFTVSTNYMSWVR chain aa QAPGKGLEWVSILYAGGVTRYADSVKTRFTISRDNSKNT LFLQMNALSAEDTAIYYCAKHYDSGYSTIDHFDSWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSQLQLVQSGTEVKKP GASVKVSCKSSGYVFTSYYLVWVRQAPGQGLEWMATISP GDVNTSYEQRFQGRVTVTTDASTNTVDMELRSLRSEDT AVYYCARGPRSKPPYLYFALDVWGQGTAVTVSSGGGGS GGGGSGGGGSEIVLTQSPGTLSLSPGETAILSCRASQSVSS SLLAWYQQKPGQAPRLLIYGASNRATGIRGRFSGSGSGT DFTLTISRLEPEDFVLYYCQHYGSRVTFGQGTKLEIK 170 Bs3GC1b heavy ggggtgcaactggtgcagtctgggggaggcttggtccagccgggggggtccctgaga chain nucl ctctcctgtgcagcctctGGATTCACCGTCAGTACCAACTACatga gctgggtccgccaggctccagggaaggggctggagtgggtctcaattCTTTAT GCCGGAGGTGTCACAaggtacgcagactccgtgaagaccagattcaccat ctccagagacaattccaagaacactctctttcttcaaatgaacgccctgagcgccgaggac acggctatatattactgtGCGAAACACTATGATTCGGGATATTC TACCATAGATCACTTTGACTCCtggggccagggaaccctggtcacc gtctcctcaGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTG GCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGC CCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTG TGACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGG CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGAC TCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGC AGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCA CAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGC CCAAATCTTGTGACAAAACTCACACATGCCCACCGTGC CCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCT CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC CACGAGGATCCTGAAGTCAAGTTCAACTGGTACGTGG ATGGCGTCGAGGTGCATAATGCCAAGACAAAACCCCG GGAGGAACAGTACAACTCAACTTATAGAGTCGTGAGC GTCCTGACCGTGCTGCATCAGGACTGGCTGAACGGCA AAGAATACAAGTGCAAAGTGTCTAATAAGGCCCTGCCT GCTCCAATCGAGAAAACAATTAGCAAGGCAAAAGGGCA GCCCAGGGAACCTCAGGTGTACACTCTGCCTCCAAGCC GCGAGGAAATGACCAAGAACCAGGTCTCCCTGACATG TCTGGTGAAAGGATTCTATCCTAGTGACATTGCCGTG GAGTGGGAATCAAATGGCCAGCCAGAGAACAATTACA AGACCACACCCCCTGTGCTGGACTCTGATGGGAGTTT CTTTCTGTATTCCAAGCTGACCGTGGATAAATCTAGA TGGCAGCAGGGAAATGTCTTTAGCTGTTCCGTGATGC ATGAGGCACTGCACAACCATTACACCCAGAAATCACTG TCACTGTCCCCAGGAAAAGGCGGGGGAGGCTCTCAGC TGCAGCTGGTCCAGAGCGGAACCGAAGTGAAGAAACC CGGCGCAAGCGTCAAAGTCTCATGCAAATCAAGCGGA TACGTCTTCACCTCTTACTATCTGGTGTGGGTCCGGC AGGCACCAGGACAGGGACTGGAGTGGATGGCCACAAT CTCTCCCGGAGACGTGAACACTAGTTACGAACAGCGA TTCCAGGGCAGAGTGACCGTCACCACAGACGCTTCAAC TAATACCGTGGATATGGAGCTGCGGAGCCTGAGATCC GAAGATACAGCCGTCTACTATTGCGCTAGGGGGCCCC GCAGCAAGCCTCCTTATCTGTATTTCGCTCTGGATG TCTGGGGGCAGGGAACAGCAGTCACCGTCTCAAGCGG CGGGGGAGGCTCTGGGGGAGGCGGGAGTGGAGGCGG GGGATCAGAGATTGTCCTGACCCAGTCACCTGGCACCC TGAGCCTGAGTCCTGGAGAGACCGCTATTCTGTCTTG TCGGGCATCACAGTCCGTGAGCTCCTCTCTGCTGGCA TGGTACCAGCAGAAGCCCGGACAGGCCCCTAGGCTGC TGATCTATGGCGCCTCCAACCGCGCTACTGGCATTCG GGGGAGATTCAGTGGCTCAGGGAGCGGAACCGACTTT ACCCTGACAATCAGCCGGCTGGAGCCCGAAGATTTCGT GCTGTACTATTGTCAGCATTATGGAAGCAGGGTCAC CTTCGGACAGGGAACTAAACTGGAAATCAAG 171 Bs3GC2b heavy EVQLVESGGDLVKAGGSLRLSCAVSGLSFSSSGMNWVRQ chain aa APGKGLEWISSISGSQNYKYYADSVKGRFVVSRDNARNFL YLQMDSLRAEDTAVYFCVGGFPYWLPPSDFSGFHVWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGKGGGGSQLQLVQSGTEV KKPGASVKVSCKSSGYVFTSYYLVWVRQAPGQGLEWMA TISPGDVNTSYEQRFQGRVTVTTDASTNTVDMELRSLRS EDTAVYYCARGPRSKPPYLYFALDVWGQGTAVTVSSGG GGSGGGGSGGGGSEIVLTQSPGTLSLSPGETAILSCRASQS VSSSLLAWYQQKPGQAPRLLIYGASNRATGIRGRFSGSGS GTDFTLTISRLEPEDFVLYYCQHYGSRVTFGQGTKLEIK 172 Bs3GC2b heavy GAGGTACAATTGGTGGAGTCTGGGGGAGACCTGGTC chain nucl AAGGCGGGGGGGTCCCTGAGACTCTCCTGTGCCGTCT CTGGATTGTCCTTCAGTAGTTCAGGCATGAATTGGGT CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCTCA TCGATTAGTGGTAGTCAGAACTACAAATACTATGCAG ACTCAGTGAAGGGCCGATTCGTCGTCTCCAGAGACAAC GCCCGCAACTTTCTATATCTGCAAATGGACAGCCTGAG GGCCGAGGATACGGCTGTGTATTTTTGTGTGGGAGG TTTCCCCTATTGGTTACCCCCGAGCGACTTCTCCGGTT TCCATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC TCAGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGC ACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGT GACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACT CTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA GCTTGGGCACCCAGACCTACATCTGCAACGTGAATCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCC CAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCC ACGAGGATCCTGAAGTCAAGTTCAACTGGTACGTGGA TGGCGTCGAGGTGCATAATGCCAAGACAAAACCCCGG GAGGAACAGTACAACTCAACTTATAGAGTCGTGAGCG TCCTGACCGTGCTGCATCAGGACTGGCTGAACGGCAA AGAATACAAGTGCAAAGTGTCTAATAAGGCCCTGCCT GCTCCAATCGAGAAAACAATTAGCAAGGCAAAAGGGCA GCCCAGGGAACCTCAGGTGTACACTCTGCCTCCAAGCC GCGAGGAAATGACCAAGAACCAGGTCTCCCTGACATG TCTGGTGAAAGGATTCTATCCTAGTGACATTGCCGTG GAGTGGGAATCAAATGGCCAGCCAGAGAACAATTACA AGACCACACCCCCTGTGCTGGACTCTGATGGGAGTTT CTTTCTGTATTCCAAGCTGACCGTGGATAAATCTAGA TGGCAGCAGGGAAATGTCTTTAGCTGTTCCGTGATGC ATGAGGCACTGCACAACCATTACACCCAGAAATCACTG TCACTGTCCCCAGGAAAAGGCGGGGGAGGCTCTCAGC TGCAGCTGGTCCAGAGCGGAACCGAAGTGAAGAAACC CGGCGCAAGCGTCAAAGTCTCATGCAAATCAAGCGGA TACGTCTTCACCTCTTACTATCTGGTGTGGGTCCGGC AGGCACCAGGACAGGGACTGGAGTGGATGGCCACAAT CTCTCCCGGAGACGTGAACACTAGTTACGAACAGCGA TTCCAGGGCAGAGTGACCGTCACCACAGACGCTTCAAC TAATACCGTGGATATGGAGCTGCGGAGCCTGAGATCC GAAGATACAGCCGTCTACTATTGCGCTAGGGGGCCCC GCAGCAAGCCTCCTTATCTGTATTTCGCTCTGGATG TCTGGGGGCAGGGAACAGCAGTCACCGTCTCAAGCGG CGGGGGAGGCTCTGGGGGAGGCGGGAGTGGAGGCGG GGGATCAGAGATTGTCCTGACCCAGTCACCTGGCACCC TGAGCCTGAGTCCTGGAGAGACCGCTATTCTGTCTTG TCGGGCATCACAGTCCGTGAGCTCCTCTCTGCTGGCA TGGTACCAGCAGAAGCCCGGACAGGCCCCTAGGCTGC TGATCTATGGCGCCTCCAACCGCGCTACTGGCATTCG GGGGAGATTCAGTGGCTCAGGGAGCGGAACCGACTTT ACCCTGACAATCAGCCGGCTGGAGCCCGAAGATTTCGT GCTGTACTATTGTCAGCATTATGGAAGCAGGGTCAC CTTCGGACAGGGAACTAAACTGGAAATCAAG 173 Bs3GC3b heavy EVQLVESGGDLVKAGGSLRLSCAVSGLSFSSSGMNWVRQ chain aa APGKGLEWISSISGSQNYKYYADSVKGRFVVSRDNARNFL YLQMDSLRAEDTAVYFCVGGFPYWLPPSDFSGFHVWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGKGGGGSGVQLVQSGGGL VQPGGSLRLSCAASGFTVSTNYMSWVRQAPGKGLEWVSI LYAGGVTRYADSVKTRFTISRDNSKNTLFLQMNALSAED TAIYYCAKHYDSGYSTIDHFDSWGQGTLVTVSSGGGGSG GGGSGGGGSDIQMTQSPDSVAVSLGERATINCKSSQSVFY TSKNKNYLAWFQQKPGQPPKLLIYWASTRESGVPDRFSG SGSGTDFTLTISSLRPEDVAVYYCQQYYSTPFTFGPGTKV DIK 174 Bs3GC3b heavy GAGGTACAATTGGTGGAGTCTGGGGGAGACCTGGTC chain nucl AAGGCGGGGGGGTCCCTGAGACTCTCCTGTGCCGTCT CTGGATTGTCCTTCAGTAGTTCAGGCATGAATTGGGT CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCTCA TCGATTAGTGGTAGTCAGAACTACAAATACTATGCAG ACTCAGTGAAGGGCCGATTCGTCGTCTCCAGAGACAAC GCCCGCAACTTTCTATATCTGCAAATGGACAGCCTGAG GGCCGAGGATACGGCTGTGTATTTTTGTGTGGGAGG TTTCCCCTATTGGTTACCCCCGAGCGACTTCTCCGGTT TCCATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC TCAGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGC ACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCTGT GACGGTCTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACT CTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA GCTTGGGCACCCAGACCTACATCTGCAACGTGAATCAC AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC CAAATCTTGTGACAAAACTCACACATGCCCACCGTGCC CAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCC ACGAGGATCCTGAAGTCAAGTTCAACTGGTACGTGGA TGGCGTCGAGGTGCATAATGCCAAGACAAAACCCCGG GAGGAACAGTACAACTCAACTTATAGAGTCGTGAGCG TCCTGACCGTGCTGCATCAGGACTGGCTGAACGGCAA AGAATACAAGTGCAAAGTGTCTAATAAGGCCCTGCCT GCTCCAATCGAGAAAACAATTAGCAAGGCAAAAGGGCA GCCCAGGGAACCTCAGGTGTACACTCTGCCTCCAAGCC GCGAGGAAATGACCAAGAACCAGGTCTCCCTGACATG TCTGGTGAAAGGATTCTATCCTAGTGACATTGCCGTG GAGTGGGAATCAAATGGCCAGCCAGAGAACAATTACA AGACCACACCCCCTGTGCTGGACTCTGATGGGAGTTT CTTTCTGTATTCCAAGCTGACCGTGGATAAATCTAGA TGGCAGCAGGGAAATGTCTTTAGCTGTTCCGTGATGC ATGAGGCACTGCACAACCATTACACCCAGAAATCACTG TCACTGTCCCCAGGAAAAGGCGGGGGAGGCTCTGGCG TGCAGCTGGTCCAGAGCGGAGGCGGACTGGTCCAGCC CGGCGGATCACTGAGACTGTCATGTGCCGCAAGCGGG TTTACCGTCTCTACAAACTACATGTCTTGGGTGAGGC AGGCACCTGGAAAGGGACTGGAGTGGGTCTCAATCCT GTACGCTGGCGGGGTGACCCGGTATGCAGACAGCGT CAAGACCCGGTTCACAATTAGCAGAGATAACTCCAAAA ATACTCTGTTTCTGCAGATGAATGCCCTGTCCGCTGA AGACACCGCAATCTACTATTGCGCCAAACACTATGATA GTGGGTACAGTACCATTGACCATTTCGATAGCTGGG GGCAGGGGACTCTGGTGACCGTCTCATCAGGCGGGGG AGGCTCTGGGGGAGGCGGGAGTGGAGGCGGGGGATC AGATATTCAGATGACCCAGAGTCCTGATTCCGTCGCT GTCTCACTGGGAGAAAGGGCAACCATTAACTGTAAAA GCTCACAGAGTGTCTTCTACACCAGTAAGAACAAAAA CTATCTGGCCTGGTTTCAGCAGAAGCCAGGCCAGCCCC CTAAACTGCTGATCTACTGGGCTAGCACTAGAGAGTC TGGAGTGCCAGACAGATTCTCTGGCAGTGGGTCAGGA ACCGACTTCACCCTGACAATTAGCTCCCTGAGGCCCGA AGACGTGGCCGTCTACTATTGTCAGCAGTATTATTCA ACACCCTTCACATTCGGACCAGGAACAAAAGTGGATA TTAAG 175 Bs3GC4 heavy QVQLMESGGGVVQPGRSLRLSCSAFGFTFSNYPMHWVR chain aa QAPGKGLEWVAIILPDGNRKNYGRSVTGRFTISRDNSNN SLYLQMNNLTTEDTAMYYCTRDGTYYSNGGVYQTYRR FFDFWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSQLQLV QSGTEVKKPGASVKVSCKSSGYVFTSYYLVWVRQAPGQG LEWMATISPGDVNTSYEQRFQGRVTVTTDASTNTVDME LRSLRSEDTAVYYCARGPRSKPPYLYFALDVWGQGTAVT VSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGETAILS CRASQSVSSSLLAWYQQKPGQAPRLLIYGASNRATGIRGR FSGSGSGTDFTLTISRLEPEDFVLYYCQHYGSRVTFGQGT KLEIK 176 BsGC4 heavy CAGGTGCAATTGATGGAGTCTGGGGGAGGCGTGGTC chain nucl CAGCCTGGGAGGTCCCTGCGACTCTCATGCAGTGCCT TTGGATTCACCTTTTCGAACTATCCTATGCACTGGGT CCGCCAGGCTCCAGGCAAGGGACTTGAGTGGGTGGCT ATCATTTTACCTGATGGGAACAGAAAAAACTATGGAA GGTCCGTGACGGGCCGATTCACCATCTCCAGAGACAAT TCCAACAACAGCCTTTATTTGCAAATGAACAACCTGAC GACTGAGGACACGGCTATGTACTATTGTACGAGAGAT GGCACGTATTACTCTAATGGTGGTGTTTATCAGACAT ATCGAAGGTTCTTCGATTTCTGGGGCCGTGGCACCCT GGTCACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGG TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGG GGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACT TCCCCGAACCTGTGACGGTCTCGTGGAACTCAGGCGCC CTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC AACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA GAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCG TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGG TGGACGTGAGCCACGAGGATCCTGAAGTCAAGTTCAA CTGGTACGTGGATGGCGTCGAGGTGCATAATGCCAAG ACAAAACCCCGGGAGGAACAGTACAACTCAACTTATAG AGTCGTGAGCGTCCTGACCGTGCTGCATCAGGACTGG CTGAACGGCAAAGAATACAAGTGCAAAGTGTCTAATA AGGCCCTGCCTGCTCCAATCGAGAAAACAATTAGCAAG GCAAAAGGGCAGCCCAGGGAACCTCAGGTGTACACTC TGCCTCCAAGCCGCGAGGAAATGACCAAGAACCAGGTC TCCCTGACATGTCTGGTGAAAGGATTCTATCCTAGTG ACATTGCCGTGGAGTGGGAATCAAATGGCCAGCCAGA GAACAATTACAAGACCACACCCCCTGTGCTGGACTCTG ATGGGAGTTTCTTTCTGTATTCCAAGCTGACCGTGGA TAAATCTAGATGGCAGCAGGGAAATGTCTTTAGCTGT TCCGTGATGCATGAGGCACTGCACAACCATTACACCCA GAAATCACTGTCACTGTCCCCAGGAAAAGGCGGGGG AGGCTCTCAGCTGCAGCTGGTCCAGAGCGGAACC GAAGTGAAGAAACCCGGCGCAAGCGTCAAAGTCT CATGCAAATCAAGCGGATACGTCTTCACCTCTTA CTATCTGGTGTGGGTCCGGCAGGCACCAGGACAG GGACTGGAGTGGATGGCCACAATCTCTCCCGGA GACGTGAACACTAGTTACGAACAGCGATTCCAGG GCAGAGTGACCGTCACCACAGACGCTTCAACTAAT ACCGTGGATATGGAGCTGCGGAGCCTGAGATCCG AAGATACAGCCGTCTACTATTGCGCTAGGGGGCC CCGCAGCAAGCCTCCTTATCTGTATTTCGCTCT GGATGTCTGGGGGCAGGGAACAGCAGTCACCGT CTCAAGCGGCGGGGGAGGCTCTGGGGGAGGCGG GAGTGGAGGCGGGGGATCAGAGATTGTCCTGAC CCAGTCACCTGGCACCCTGAGCCTGAGTCCTGGA GAGACCGCTATTCTGTCTTGTCGGGCATCACAGT CCGTGAGCTCCTCTCTGCTGGCATGGTACCAGCA GAAGCCCGGACAGGCCCCTAGGCTGCTGATCTAT GGCGCCTCCAACCGCGCTACTGGCATTCGGGGGA GATTCAGTGGCTCAGGGAGCGGAACCGACTTTAC CCTGACAATCAGCCGGCTGGAGCCCGAAGATTTC GTGCTGTACTATTGTCAGCATTATGGAAGCAGG GTCACCTTCGGACAGGGAACTAAACTGGAAATCA AG 177 Bs3GC5 heavy QVQLMESGGGVVQPGRSLRLSCSAFGFTFSNYPMHWVR chain aa QAPGKGLEWVAIILPDGNRKNYGRSVTGRFTISRDNSNN SLYLQMNNLTTEDTAMYYCTRDGTYYSNGGVYQTYRR FFDFWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGVQLV QSGGGLVQPGGSLRLSCAASGFTVSTNYMSWVRQAPGK GLEWVSILYAGGVTRYADSVKTRFTISRDNSKNTLFLQM NALSAEDTAIYYCAKHYDSGYSTIDHFDSWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPDSVAVSLGERATINC KSSQSVFYTSKNKNYLAWFQQKPGQPPKLLIYWASTRES GVPDRFSGSGSGTDFTLTISSLRPEDVAVYYCQQYYSTPF TFGPGTKVDIK 178 Bs3GC5 heavy CAGGTGCAATTGATGGAGTCTGGGGGAGGCGTG chain nucl GTCCAGCCTGGGAGGTCCCTGCGACTCTCATGCA GTGCCTTTGGATTCACCTTTTCGAACTATCCTAT GCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTT GAGTGGGTGGCTATCATTTTACCTGATGGGAACA GAAAAAACTATGGAAGGTCCGTGACGGGCCGATT CACCATCTCCAGAGACAATTCCAACAACAGCCTTT ATTTGCAAATGAACAACCTGACGACTGAGGACAC GGCTATGTACTATTGTACGAGAGATGGCACGTAT TACTCTAATGGTGGTGTTTATCAGACATATCGAA GGTTCTTCGATTTCTGGGGCCGTGGCACCCTGGT CACCGTCTCCTCAGCGTCGACCAAGGGCCCATCGG TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCT GGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGG ACTACTTCCCCGAACCTGTGACGGTCTCGTGGAAC TCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC GGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA GCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGG CACCCAGACCTACATCTGCAACGTGAATCACAAGC CCAGCAACACCAAGGTGGACAAGAGAGTTGAGCC CAAATCTTGTGACAAAACTCACACATGCCCACCGT GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT GATCTCCCGGACCCCTGAGGTCACATGCGTGGTG GTGGACGTGAGCCACGAGGATCCTGAAGTCAAGT TCAACTGGTACGTGGATGGCGTCGAGGTGCATAA TGCCAAGACAAAACCCCGGGAGGAACAGTACAACT CAACTTATAGAGTCGTGAGCGTCCTGACCGTGCT GCATCAGGACTGGCTGAACGGCAAAGAATACAAG TGCAAAGTGTCTAATAAGGCCCTGCCTGCTCCAA TCGAGAAAACAATTAGCAAGGCAAAAGGGCAGCC CAGGGAACCTCAGGTGTACACTCTGCCTCCAAGCC GCGAGGAAATGACCAAGAACCAGGTCTCCCTGAC ATGTCTGGTGAAAGGATTCTATCCTAGTGACATT GCCGTGGAGTGGGAATCAAATGGCCAGCCAGAGA ACAATTACAAGACCACACCCCCTGTGCTGGACTCT GATGGGAGTTTCTTTCTGTATTCCAAGCTGACCG TGGATAAATCTAGATGGCAGCAGGGAAATGTCTT TAGCTGTTCCGTGATGCATGAGGCACTGCACAAC CATTACACCCAGAAATCACTGTCACTGTCCCCAGG AAAAGGCGGGGGAGGCTCTGGCGTGCAGCTGGTCCA GAGCGGAGGCGGACTGGTCCAGCCCGGCGGATCACTG AGACTGTCATGTGCCGCAAGCGGGTTTACCGTCTCTA CAAACTACATGTCTTGGGTGAGGCAGGCACCTGGAAA GGGACTGGAGTGGGTCTCAATCCTGTACGCTGGCGG GGTGACCCGGTATGCAGACAGCGTCAAGACCCGGTTC ACAATTAGCAGAGATAACTCCAAAAATACTCTGTTTCT GCAGATGAATGCCCTGTCCGCTGAAGACACCGCAATCT ACTATTGCGCCAAACACTATGATAGTGGGTACAGTA CCATTGACCATTTCGATAGCTGGGGGCAGGGGACTC TGGTGACCGTCTCATCAGGCGGGGGAGGCTCTGGGG GAGGCGGGAGTGGAGGCGGGGGATCAGATATTCAGA TGACCCAGAGTCCTGATTCCGTCGCTGTCTCACTGGG AGAAAGGGCAACCATTAACTGTAAAAGCTCACAGAGT GTCTTCTACACCAGTAAGAACAAAAACTATCTGGCCT GGTTTCAGCAGAAGCCAGGCCAGCCCCCTAAACTGCTG ATCTACTGGGCTAGCACTAGAGAGTCTGGAGTGCCAG ACAGATTCTCTGGCAGTGGGTCAGGAACCGACTTCAC CCTGACAATTAGCTCCCTGAGGCCCGAAGACGTGGCCG TCTACTATTGTCAGCAGTATTATTCAACACCCTTCAC ATTCGGACCAGGAACAAAAGTGGATATTAAG Non-engineered chains of multispecific antibodies 179 GCA7 heavy chain GVQLVQSGGGLVQPGGSLRLSCAASgftvs t nyMSWVRQAP aa GKGLEWVSIl y a gg v t RYADSVKTRFTISRDNSKNTLFLQM NALSAEDTAIYYCakhy d sgystidhfd s WGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 180 GCA7 heavy chain ggggtgcaactggtgcagtctgggggaggcttggtccagccgggggggtccctgaga nucl ctctcctgtgcagcctctGGATTCACCGTCAGTACCAACTACatga gctgggtccgccaggctccagggaaggggctggagtgggtctcaattCTTTAT GCCGGAGGTGTCACAaggtacgcagactccgtgaagaccagattcaccat ctccagagacaattccaagaacactctctttcttcaaatgaacgccctgagcgccgaggac acggctatatattactgtGCGAAACACTATGATTCGGGATATTC TACCATAGATCACTTTGACTCCtggggccagggaaccctggtcacc gtctcctcagcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagca cctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaacctgtg acggtctcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtccta cagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggc acccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaga gttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcct ggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccgg acccctgaggtcacatgcgtggtggtggacgtgagccacgaGgaTcctgaggtcaag ttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggagg agcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggc tgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgaga aaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccat cccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatc ccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagac cacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggaca agagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcac aaccactacacgcagaagagcctctccctgtccccgggtaaa 181 GCA7 light chain DIQMTQSPDSVAVSLGERATINCKSSqsv f y t s k nknyLAWFQ aa QKPGQPPKLLIYwasTRESGVPDRFSGSGSGTDFTLTISSL RPEDVAVYYCqqyystpftFGPGTKVDIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 182 GCA7 light chain gacatccagatgacccagtctccagactccgtggctgtgtctctgggcgagagggccacc nucl atcaactgcaagtccagcCAGAGTGTTTTCTACACCTCCAAAAA TAAAAACTACttagcttggttccagcagaaaccaggacagcctcctaaactgctc atttacTGGGCATCTacccgggagtccggggtccctgaccgattcagtggcag cgggtctgggacagatttcactctcaccatcagcagcctgcggcctgaagatgtggcagt ttattactgtCAGCAATATTATAGTACCCCTTTCACTttcggccc tgggaccaaagtggatatcaaacgTacGgtggctgcaccatctgtcttcatcttcccgcc atctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatc ccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccag gagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgac gctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagg gcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 183 GCA21 light chain DIQMTQSPSTLSTSVGDRVTITCRASq n il n wLAWYQQKPG aa NAPNLLIYkas DLQSGVPSRFSGSGSGTEFTLTISSLQPDDF ATYYCq h ynsy p ltFGGGTKVEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 184 GCA21 light chain gacatccagatgacccagtctccttccaccctgtctacatctgtgggagacagagtcaccat nucl cacttgccgggccagtCAGAATATCCTTAATTGGttggcctggtatcaa cagaaaccagggaacgcccctaacctcctgatatatAAGGCGTCTgatttacaaa gtggggtcccctcaagattcagcggcagtgggtctgggacagaattcactctcaccatca gcagcctgcagcctgatgattttgcaacttattactgcCAGCATTATAATAG TTATCCTCTCACTttcggcggagggaccaaggtggaaatcaaacgTacG gtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgc ctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtg gataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaagga cagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaa agtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaa caggggagagtgt 185 GCB59 light chain SYVLTQPPSVSVAPGQTASLTCGGT NIGSKSVHWYQQKA aa GQAPVLVVY A D N DRPSGVPERFSGSNSGNTATLTISRVEA EDESDYFCQVWD GNT DHVVFGGGTKLTVLGQPKAAPS VTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS 186 GCB59 light chain TCATATGTGCTGACTCAACCACCCTCGGTGTCAGTGGC nucl CCCAGGACAGACGGCCAGTCTAACCTGTGGGGGAACT AACATTGGAAGTAAAAGTGTTCATTGGTACCAGCAAA AGGCAGGCCAGGCCCCTGTGTTGGTCGTCTATGCTGA TAACGACAGGCCCTCAGGGGTCCCTGAGCGATTCTCT GGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAG CAGGGTCGAGGCCGAGGATGAGTCCGACTATTTCTGT CAGGTGTGGGATGGTAATACTGATCATGTGGTCTT CGGCGGAGGGACCAAGCTGACCGTCCTGggtcagcccaaggct gccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacact ggtgtgtctcataagtgacttctacccgggagccgtgacagtggcttggaaagcagata gcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaag tacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagcta cagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaa tgttca 187 GCE536 heavy QLQLVQSGTEVKKPGASVKVSCKSSGY V FTSYY LVWVRQ chain aa APGQGLEWMAT I S P GDVN TSYEQRFQGRVTVTTDASTN TVDMELRSLRSEDTAVYYCARGPRSK PP YLYF AL DVWG QGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 188 GCE536 heavy CAGCTGCAGCTGGTCCAGTCAGGCACAGAGGTCAAAA chain nucl AGCCAGGAGCATCAGTGAAGGTGTCTTGTAAGTCATC AGGATACGTGTTCACCTCTTACTATCTGGTGTGGGT CCGGCAGGCACCAGGACAGGGACTGGAGTGGATGGCC ACAATCTCTCCCGGAGACGTGAACACTAGTTACGAAC AGCGATTCCAGGGCAGAGTGACCGTCACCACAGACGCT TCAACTAATACCGTGGATATGGAGCTGCGGAGCCTGA GATCCGAAGATACAGCCGTCTACTATTGCGCTAGGGG GCCCCGCAGCAAGCCTCCTTATCTGTATTTTGCTCT GGATGTGTGGGGGCAGGGGACCGCTGTCACCGTGTC AAGCgcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacc tctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaacctgtgac ggtctcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctaca gtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacc cagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagtt gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgg ggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacc cctgaggtcacatgcgtggtggtggacgtgagccacgaGgaTcctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggag cagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctg aatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaa accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatccc gggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatccc agcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagacca cgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaa gagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcaca accactacacgcagaagagcctctccctgtccccgggtaaa 189 GCE536 light EIVLTQSPGTLSLSPGETAILSCRASQSVSSS L LAWYQQKP chain aa GQAPRLLIYGAS NRATGIRGRFSGSGSGTDFTLTISRLEPE DFVLYYCQ H YGSRVTFGQGTKLEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 190 light GCE536 gaaattgtgttgacgcagtctcctggcaccctgtctttgtctccaggggaaacagccatcct chain nucl ctcctgcagggccagtcagagtgtcagcagcagcctcttagcctggtaccagcaaaaa cctggccaggctcccaggctcctcatctacggtgcatccaatagggccactggcatcaga ggcaggtttagtggcagtgggtctgggacagacttcactctcaccatcagtagattggag cctgaagattttgtactttattactgtcagcactatggctcacgggtcacttttggccag gggaccaagctggagatcaaacgTacGgtggctgcaccatctgtcttcatcttcccgcc atctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatc ccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccag gagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgac gctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagg gcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt *the sequences highlighted in bold are CDR regions (nucleotide or aa) and the underlined residues are mutated residues as compared to the ″germline″ sequence. 

1. A method for prophylaxis, treatment or attenuation of an inflammatory and/or an autoimmune disease, comprising administering to the subject an effective amount of a multispecific anti-cytokine antibody, or an antigen binding fragment thereof, comprising: (a) at least two epitope binding sites, wherein each of the at least two epitope binding sites is specific for a different epitope of a cytokine, whereby the different epitopes of the cytokine are non-overlapping epitopes; and (b) an Fc moiety.
 2. The method according claim 1, wherein the antibody, or the antigen binding fragment thereof, is bispecific, trispecific, tetraspecific or pentaspecific.
 3. The method according to claim 1, wherein the antibody, or the antigen binding fragment thereof, comprises two different epitope binding sites specific for at least two different, non-overlapping epitopes of the cytokine.
 4. The method according to claim 1, wherein the anti-cytokine antibody, or the antigen binding fragment thereof, is selected from the group consisting of an anti-colony stimulating factor antibody and an anti-interferon antibody, preferably the anti-cytokine antibody is an anti-GM-CSF antibody or an anti-interferon beta antibody.
 5. The method according to claim 1, wherein the anti-cytokine antibody, or the antigen binding fragment thereof, is an anti-GM-CSF antibody.
 6. The method according to claim 5, wherein the antibody, or the antigen binding fragment thereof, is a bispecific tetravalent antibody or a trispecific hexavalent antibody.
 7. The method according to claim 1, wherein the antibody, or the antigen binding fragment thereof, further comprises: (c) at least one linker.
 8. The method according to claim 7, wherein the linker comprises or consists of an amino acid sequence according to SEQ ID NO: 143 or SEQ ID NO: 144 or a functional sequence variant thereof.
 9. The method according to claim 1, wherein the antibody, or the antigen binding fragment thereof, comprises: (a) an IgG type, (b) an IgG1 type, (c) a heavy chain constant region of the IgG1 CH1-CH2-CH3 type and a light chain constant region of the IgG CK type, or (d) a heavy chain constant region of the IgG1 CH1-CH2-CH3 type comprising or consisting of an amino acid sequence according to SEQ ID NO: 140 or functional sequence variants thereof, and a light chain constant region of the IgG CK type comprising or consisting of an amino acid sequence according to SEQ ID NO: 141 or functional sequence variants thereof.
 10. The method according to claim 1, wherein the antibody, or the antigen binding fragment thereof, is of a construct type selected from the group comprising Bs1, Bs2, Bs3, Ts1, Ts2 and Ts3.
 11. The method according to claim 10, wherein the antibody, or the antigen binding fragment thereof, comprises a construct type Ts3 or comprises a trispecific antibody according to the construct type Ts3.
 12. The method according to claim 1, wherein the antibody, or the antigen binding fragment thereof, is a human antibody, a monoclonal antibody, a human monoclonal antibody, a purified antibody, or a single chain antibody, or an antigen binding fragment thereof.
 13. The method according to claim 1, wherein the antibody, or the antigen binding fragment thereof, neutralizes the cytokine: (i) under stringent conditions with an IC₉₀ of 150 ng/ml or less; (ii) under less stringent conditions with an IC₉₀ of 20 ng/ml or less; (iii) under more stringent conditions with an IC₉₀ of 160 ng/ml or less; and/or (iv) under very stringent conditions with an IC₉₀ of 1000 ng/ml or less.
 14. The method according to claim 1, wherein the antibody or antigen binding fragment comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein the at least one heavy chain CDRH3 comprises an amino acid sequence according to SEQ ID NOs: 3, 51, 69, or 107 or functional sequence variants thereof, or the at least one heavy chain CDRH3 comprises an amino acid sequence according to SEQ ID NOs: 3 or 69 or functional sequence variants thereof.
 15. The method according to claim 1, wherein the antibody or antigen binding fragment comprises: (i) CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 1-5 and 7 or functional sequence variants thereof or according to SEQ ID NOs: 1-4 and 6-7 or functional sequence variants thereof, respectively; (ii) CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 49-53 and 55 or functional sequence variants thereof or according to SEQ ID NOs: 49-52 and 54-55 or functional sequence variants thereof, respectively; (iii) CDRH1, CDRH2, and CDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 67-71 and 73 or functional sequence variants thereof or according to SEQ ID NOs: 67-70 and 72-73 or functional sequence variants thereof, respectively; and/or (iv) heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs: 105-109 and 111 or functional sequence variants thereof or according to SEQ ID NOs: 105-108 and 110-111 or functional sequence variants thereof, respectively.
 16. The method according to claim 1, wherein the antibody or antigen binding fragment comprises: (i) a VH amino acid sequence according to SEQ ID NO: 37 or functional sequence variants thereof and a VL amino acid sequence according to SEQ ID NO: 38 or functional sequence variants thereof; (ii) a VH amino acid sequence according to SEQ ID NO: 63 or functional sequence variants thereof and a VL amino acid sequence according to SEQ ID NO: 64 or functional sequence variants thereof; (iii) a VH amino acid sequence according to SEQ ID NO: 95 or functional sequence variants thereof and a VL amino acid sequence according to SEQ ID NO: 96 or functional sequence variants thereof; and/or (iv) a VH amino acid sequence according to SEQ ID NO: 130 or functional sequence variants thereof and a VL amino acid sequence according to SEQ ID NO: 131 or functional sequence variants thereof.
 17. The method according to claim 1, wherein the heavy chain of the antibody or antigen binding fragment comprises a CDRL1 amino acid sequence selected from the amino acids sequences according to SEQ ID NOs: 4, 52, 70, 108 or functional sequence variants thereof, a CDRL2 amino acid sequence selected from the amino acids sequences according to SEQ ID NOs: 5, 6, 53, 54, 71, 72, 109, 110 or functional sequence variants thereof, and/or a CDRL3 amino acid sequence selected from the amino acids sequences according to SEQ ID NOs: 7, 55, 73, 111 or functional sequence variants thereof.
 18. The method according to claim 1, wherein the heavy chain of the antibody or antigen binding fragment comprises a VL amino acid sequence selected from the amino acids sequences according to SEQ ID NOs: 38, 64, 96, 131 or functional sequence variants thereof; or the heavy chain of the antibody or antigen binding fragment comprises a VL amino acid sequence according to SEQ ID NOs: 38 or 96 or functional sequence variants thereof; or the heavy chain of the antibody or antigen binding fragment comprises a VL amino acid sequence according to SEQ ID NO: 96 or functional sequence variants thereof.
 19. The method according to claim 1, wherein: (i) the antibody or antigen binding fragment is of a construct type select from the group consisting of the construct types Bs1, Bs2, Bs3, Ts1, Ts2 and Ts3; and (ii) comprises at any of the positions A and/or C a CDRH1 amino acid sequence, a CDRH2 amino acid sequence, a CDRH3 amino acid sequence, a CDRL1 amino acid sequence, a CDRL2 amino acid sequence and a CDRL3 amino acid sequence selected from the group consisting of amino acid sequences according to SEQ ID NOs: 1-7 and 67-73 or functional sequence variants thereof; preferably comprises at position A a CDRH1 amino acid sequence, a CDRH2 amino acid sequence, a CDRH3 amino acid sequence, a CDRL1 amino acid sequence, a CDRL2 amino acid sequence and a CDRL3 amino acid sequence according to SEQ ID NOs: 67-71 and 73 or functional sequence variants thereof or according to SEQ ID NOs: 67-70 and 72-73 or functional sequence variants thereof.
 20. The method according to claim 1, wherein the antibody, or the antigen binding fragment thereof, is according to gTs1GC1, gTs1GC2a, gTs2GC2b, gTs2GC2c, gTs3GC2d, gTs3GC2e, gBs3GC1a, gBs3GC1b, gBs2GC1c, gBs2GC1d, gBs1GC2a, gBs3GC2b, gBs1GC3a, gBs3GC3b, gBs3GC4, or gBs3GC5.
 21. The method according to claim 20, wherein the antibody, or the antigen binding fragment thereof, is Ts1GC1, Ts1GC2a, Ts2GC2b, Ts2GC2c, Ts3GC2d, Ts3GC2e, Bs3GC1a, Bs3GC1b, Bs2GC1c, Bs2GC1d, Bs1GC2a, Bs3GC2b, Bs1GC3a, Bs3GC3b, Bs3GC4, or Bs3GC5.
 22. The method according to claim 1, wherein the inflammatory disease is due to a physical cause, an immune reaction due to hypersensitivity, a chemical cause, or is elected from the group consisting of appendicitis, bursitis, colitis, cystitis, dermatitis, phlebitis, RSD/CRPS, rhinitis, tendonitis, tonsillitis, vasculitis, Alzheimer's disease, ankylosing spondylitis, arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis, asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematous (SLE), nephritis, Parkinson's disease, ulcerative colitis, Acne vulgaris, autoinflammatory diseases, celiac disease, prostatitis, pulmonary alveolar proteinosis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, vasculitis, interstitial cystitis, inflammatory myopathies, encephalomyelitis, in particular acute disseminated encephalomyelitis, spondylitis, in particular ankylosing spondylitis, antisynthetase syndrome, dermatitis, in particular atopic * dermatitis or contact dermatitis, hepatitis, in particular autoimmune hepatitis, autoimmune peripheral neuropathy, pancreatitis, in particular autoimmune pancreatitis, Behet's disease, Bickerstaffs, encephalitis, Blau syndrome, Coeliac disease, Chagas disease, polyneuropathy, in particular chronic inflammatory demyelinating polyneuropathy, osteomyelitis, in particular chronic recurrent multifocal osteomyelitis, Churg-Strauss syndrome, Cogan syndrome, giant-cell arteritis, CREST syndrome, vasculitis, in particular cutaneous small-vessel vasculitis and urticarial vasculitis, dermatitis herpetiformis, dermatomyositis, systemic scleroderma, Dressler's syndrome, drug-induced lupus erythematosus, discoid lupus erythematosus, enthesitis, eosinophilic fasciitis, eosinophilic gastroenteritis, erythema nodosum, Idiopathic pulmonary fibrosis, gastritis, Grave's disease, Guillain-barre syndrome, Hashimoto's thyroiditis, Henoch-Schonlein purpura, hydradenitis suppurativa, Idiopathic inflammatory demyelinating diseases, myositis, in particular inclusion body myositis, cystitis, in particular interstitial cystitis, Kawasaki disease, Lichen planus, lupoid hepatitis, Majeed syndrome, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, myelitis, in particular neuromyelitis optica, thyroiditis, in particular Ord's thyroiditis, rheumatism, in particular palindromic rheumatism, Parsonage-Turner syndrome, pemphigus vulgaris, perivenous encephalomyelitis, polyarteritis nodosa, polymyalgia, in particular polymyalgia rheumatica, polymyositis, cirrhosis, in particular primary biliary cirrhosis, cholangitis, in particular primary sclerosing cholangitis, progressive inflammatory neuropathy, Rasmussen's encephalitis, relapsing polychondritis, arthritis, in particular reactive arthritis (Reiter disease) and rheumatoid arthritis, rheumatic fever, sarcoidosis, Schnitzler syndrome, serum sickness, spondyloarthropathy, Takayasu's arteritis, Tolosa-Hunt syndrome, transverse myelitis, or Wegener's granulomatosis.
 23. The method of claim 1, wherein the autoimmune disease autoimmune is a disease of the CNS, an auto-inflammatory disease, Celiac disease; Sjogren's syndrome, systemic lupus erythematosus, Blau syndrome, Bullous pemphigoid, Cancer, Castleman's disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating polyneuropathy, Chronic recurrent multifocal osteomyelitis, chronic obstructive pulmonary disease, Churg-Strauss syndrome, Cicatricial pemphigoid, Cogan syndrome, Cold agglutinin disease, Complement component 2 deficiency, Contact dermatitis, Cranial arteritis, CREST syndrome, Crohn's disease, Cushing's Syndrome, Dercum's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type 1 Diffuse cutaneous systemic sclerosis, Dressler's syndrome, lupus, Discoid lupus erythematosus, Eczema, Acute disseminated encephalomyelitis (ADEM), Addison's disease, Agammaglobulinemia, Amyotrophic lateral sclerosis (Also Lou Gehrig's disease; Motor Neuron Disease), Ankylosing Spondylitis Antiphospholipid syndrome, Anti synthetase syndrome, Atopic dermatitis, Autoimmune aplastic anemia, Autoimmune cardiomyopathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome, Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome, Autoimmune progesterone dermatitis, Autoimmune thrombocytopenic purpura, Autoimmune urticarial, Autoimmune uveitis, Balo disease/Balo concentric sclerosis, Behcet's disease, Berger's disease, Bickerstaffs encephalitis, Endometriosis, Enthesitis-related arthritis, Eosinophilic gastroenteritis, Epidermolysis bullosa acquisita, Erythroblastosis fetalis, Evan's syndrome, Fibrodysplasia ossificans, Fibrosing alveolitis (or Idiopathic pulmonary fibrosis), Gastritis, Glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's encephelopathy, Hashimoto's thyroiditis, Gestational Pemphigoid, Hidradenitis suppurativa, Hypogammaglobuli nemia, Idiopathic thrombocytopenic purpura (Autoimmune thrombocytopenic purpura), IgA nephropathy, Occular cicatricial pemphigoid, Inclusion body myositis, Rheumatoid arthritis, Chronic inflammatory Rheumatic fever, demyelinating polyneuropathy, Sarcoidosis, Palindromic rheumatism, Interstitial cystitis, Juvenile idiopathic Schizophrenia, PANDAS (pediatric arthritis aka Juvenile autoimmune rheumatoid arthritis), Schmidt syndrome, neuropsychiatric Kawasaki's disease another form of APS, Schnitzler syndrome, Paraneoplastic cerebellar myasthenic syndrome, Leukocytoclastic Serum Sickness, Lichen planus, Sjogren's syndrome, Lichen sclerosus, Parsonage-Turner, Linear IgA disease, Still's disease, Pemphigus vulgaris, Lupoid hepatitis, Autoimmune hepatitis, Stiff person syndrome, Pernicious anaemia, Subacute bacterial endocarditis (SBE), POEMS syndrome, Lupus erythematosus, Sweet's syndrome, Sympathetic ophthalmia, Meniere's disease, Systemic lupus, Primary biliary cirrhosis, Miller-Fisher syndrome, Takayasu's arteritis, cholangitis, Progressive inflammatory neuropathy, Mucha-Habermann disease, Psoriasis, Psoriatic arthritis, Pyoderma gangrenosum, Multiple sclerosis, Pure red cell aplasia, Rasmussen's encephalitis, Myasthenia gravis, Transverse myelitis, Raynaud phenomenon, Microscopic colitis, Ulcerative colitis, Myositis, idiopathic inflammatory bowel disease (IBD), Neuromyelitis optica, Devic's disease, or Neuromyotonia. 